The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.DOI:http://dx.doi.org/10.7554/eLife.23152.001 Cells use a protein called actin to provide shape, to generate the forces needed for cells to divide, and for many other essential processes. Inside a cell, individual actin proteins join up to form long filaments. These actin filaments are organized in different ways to make networks that have distinct properties, each tailored for a specific process. For instance, bundles of straight actin filaments help a cell to divide, whereas a network of branched actin filaments allows cells to move.The different proteins that bind to actin filaments influence how quickly actin filaments are assembled and organized into networks. Therefore, many of the properties of an actin filament network are due to the actin binding proteins that are associated with it. Two actin binding proteins called fimbrin and cofilin associate with a type of actin filament network known as the actin patch. A third actin binding protein called tropomyosin associates with a different network that forms a ring. It is not known how particular actin binding proteins choose to associate with one actin network instead of another.Christensen et al. used a fluorescence microscopy technique to study how fimbrin, cofilin and tropomyosin associate with different actin networks in a single-celled organism called fission yeast. This technique involved incubating actin and actin binding proteins together in a microscope chamber. The experiments show that some actin binding proteins, like tropomyosin, cooperate to bind to actin. Individual tropomyosin molecules find it difficult to bind actin filaments on their own, but once one tropomyosin molecule is attached to the filament, others rapidly join to coat the filament.On the other hand, some actin-binding proteins compete for binding to filaments. For example, the binding of fimbrin to actin filaments causes tropomyosin to be removed from the actin network. Further experiments revealed that fimbrin and cofilin work with each other to rapidly generate a dense actin network and displace tropomyosin. Together, the findings of Christensen et al. suggest that competitions between actin binding proteins determine which actin binding proteins are associated with an actin network.The next challenge is to understand how the most competitive actin-binding proteins are kept off actin networks where they do not belong. Further studies will shed light on how these interactions cause large changes in how the cell is organized.DOI:http://dx.doi.org/10.7554/eLife.23152.002 C. elegans embryo  spans four actin subunits. Although Cdc8, like other tropomyosins, has been purified and characterized in steady state bulk assays . However, there was considerable variation in spreading rates in both directions, between ~1–6 Cdc8 molecules sec−1 μM−1  the best match to the experimental data in on is varied, (2) a similar koff to single molecule experimental measurements . To further address whether indirect cooperativity plays a role in tropomyosin Cdc8 loading, we created two variations of a lattice simulation describing Cdc8 loading onto a growing actin filament . The firurements , and (3)urements . In the urements . A kymogurements . Howeverurements  comparedurements . Therefourements . An indiurements . This mourements , and alsurements . These furements , are confim1-1Δ cells)  . In addiΔ cells) . HoweverTo differentiate between these and other potential mechanisms, we utilized three-color TIRFM to follow the assembly of labeled actin in the presence of labeled Fim1 and Cdc8 to simultaneously observe their associations with F-actin . Cdc8 inADF/cofilin Adf1 is an F-actin severing protein that localizes predominantly to actin patches and is required for proper actin patch dynamics , but alson) of Adf1 with actin filaments, the dissociation rate (koff) of Adf1 from F-actin was unaffected by Cdc8 , the majority of F-actin was rapidly coated with Adf1 . However by Cdc8 . These f by Cdc8 . Initial by Cdc8 . Cdc8 co by Cdc8 , we susp of Cdc8 .10.7554/Within an actin patch, a dense array of Arp2/3 complex-mediated branched F-actin provides the network architecture that propels newly-generated endocytic vesicles inward . We prevOur findings and previous work have suggested that the synergistic activities of fimbrin Fim1 and ADF/cofilin Adf1 may serve to inhibit the association of tropomyosin Cdc8 with F-actin . We testOur work demonstrates that cooperativity is an important characteristic for multiple aspects of F-actin network formation, organization, and ABP sorting. We show that fission yeast tropomyosin Cdc8 binds extremely cooperatively to F-actin . Though BTln(2)=~0.4 kcal/mol. Hence, if this indirect cooperativity is due to a structural change in the actin filament, we expect that change to be quite subtle.End-to-end binding has been considered to be the primary source of tropomyosin’s cooperativity. Mutations in the N- or C-terminal domains of Cdc8 affect its ability to bind cooperatively and polymerize on an actin filament . HoweverTropomyosin is known to increase the persistence length of F-actin . As a reHow does this high degree of cooperativity affect tropomyosin’s influence on the activity of other ABPs? The nature of tropomyosin as a strand-like, end-to-end associated protein makes it an ideal F-actin ‘gatekeeper’ , 2015. IAnother impact of Cdc8’s high cooperativity may be that only a slight bias toward certain actin filaments could generate an ‘all-or-nothing’ sorting toward those networks. Several studies have suggested that the actin assembly factor may be the key to generating this initial bias . In fissOur work also addresses the importance of ABP competition in network organization and ABP sorting. An outstanding question is how ADF/cofilin, a protein that severs at low concentrations, can be present at high concentrations in the cell and yet still rapidly disassemble F-actin networks. Other ABPs, such as Aip1 , CoroninIn addition, our study suggests an important role for ADF/cofilin not only as a disassembly factor, but also as a potent F-actin network assembly factor through the creation of new barbed ends as in the model of dendritic nucleation within the lamellipodia of migrating cells . The comListeria comet tails  localization on the outside of the coiled coil , (2) low sequence conservation amongst fungal tropomyosins . Three amino acids in omyosins , (3) preomyosins , and (4)omyosins , , and the product was cloned into gene targeting vector pFA6-kanMX6 at BglII using In-Fusion HD Cloning . cdc8mut-kan was then amplified (using primers AGGTATGAGATGATAGCTTTTCATTGGAAAATCAAGTTGCTAATATTTGCTTTTTATTTAGAAAATTAATGCCGCTCGTGC and AGAAGATATAAAAAAGGTGGTATGTTTCTTCTATGTTCGTCAAGCTTTTCGCTATGAATTCGAGCTCGTTTAAAC) and transformed into a temperature sensitive mutant cdc8-27 strain. Colonies were screened for absence of temperature sensitivity and resistance to kanamycin. Candidate colonies were then screened for proper insertion by PCR and sequenced to confirm insertion at the cdc8 locus. Strains created are listed in All three mutations were introduced within exon 2 of the S. pombe ADF/cofilin Adf1 for labeling in TIRFM experiments. Endogenous cysteine residues were converted to alanine (C12S and C62A), and aspartate 34 was converted to cysteine (D34C) as previously described for ADF/cofilin from S. cerevisiae , and tropomyosin AlaSer-Cdc8  were expressed in BL21-Codon Plus (DE3)-RP  and purified as described previously . Briefly280 of purified proteins was taken using a Nanodrop 2000c Spectrophotometer . Protein concentration was calculated using extinction coefficients Fim1: 55,140 M−1 cm−1, Cdc8 (WT and mutants): 2980 M−1 cm−1, Adf1 (and mutant): 13,075 M−1 cm−1. Proteins were labeled with CFTM405M , TMR-6-maleimide  or Cy5-monomaleimide  dyes as per manufacturer’s protocols immediately following purification, and were flash-frozen in liquid nitrogen and kept at −80°C. Cdc8 was reduced with DTT prior to labeling. For proteins labeled on one cysteine residue (Cdc8 and Adf1 mutants), labeling efficiency was determined by taking the absorbance at the emission max of the dye and calculating the coupling efficiency  for TIRFM were prepared by washes in acetone, isopropanol, and water followed by sonication for 30 min in isopropanol. Washed glass was then cleaned by plasma cleaning for 3 min using a Harrick PDC-32G plasma cleaner . Cleaned coverslips and microscope slides were immediately passivated by incubation in 1 mg/mL PEG-Si (5000 MW) in 95% ethanol for 18 hr . Coversl2, 1 mM EGTA, 50 mM DTT, 0.2 mM ATP, 50 μM CaCl2, 15 mM glucose, 20 μg/mL catalase, 100 μg/mL glucose oxidase, and 0.5% (400 centipoise) methylcellulose) to induce F-actin assembly (Time-lapse TIRFM movies were obtained using a cellTIRF 4Line system (Olympus) fitted to an Olympus IX-71 microscope with through-the-objective TIRF illumination and a iXon EMCCD camera . Mg-ATP-actin (15% Alexa 488-labeled) was mixed with labeled or unlabeled ABPs and a polymerization mix (10 mM imidazole (pH 7.0), 50 mM KCl, 1 mM MgClassembly . The mixLine scans were performed by drawing a three pixel width line along the actin filament and recording the fluorescence intensity along the line using ‘Plot Profile’ in FIJI . The samThe occupancy of Cdc8 on actin filaments was determined from one frame from each TIRFM movie. For each movie, the frame of interest was chosen based on F-actin density (between 7 and 11 μm of filament per square μm) rather than time point. Segmented line ROIs were used to measure total actin filament length (488 channel) and total Cdc8 cable length (647 channel) at that frame. History of the TIRFM movie as well as Cdc8 fluorescence intensity was used to determine sites at which two Cdc8 cables were present on a single actin filament stretch, and in these cases each Cdc8 stretch was counted as a separate measurement. As there are two Cdc8 cables on a single actin filament, Total Cdc8 Occupancy=Total Cdc8 Length/(Actin Filament Length*2). Single vs double Cdc8 occupancy was determined by measuring the length of single- vs double-Cdc8-coated stretches and calculating the total length of single- or double-coated stretches divided by total actin filament length.n//4)*0.001 (adapted from ). 250  were used to define each actin filament in the actin channel (488 channel). ROIs were then transferred to the ABP channel of interest and mean fluorescence intensity was measured for each actin filament. For comparing single actin filaments to F-actin bundles, the history of the actin channel and the actin fluorescence intensity was used to determine single filament versus bundled regions and separate ROI sets were generated and used to measure fluorescence intensity.off.To calculate residence times for individual ADF/cofilin Adf1 or tropomyosin Cdc8 molecules, a high total concentration of Adf1 or Cdc8 was included in the TIRFM assay to ensure total coating of the actin filaments (5 μM Adf1 or 2.5 μM Cdc8).~20% of Adf1 or Cdc8 was labeled with Cy5 dye in order to visualize the extent of coating of the protein on F-actin. The actin and Cy5-labeled Adf1 or Cdc8 was visualized every 10 s. A low (0.5–1%) percentage of Adf1 or Cdc8 was labeled with TMR in order to visualize single molecules, and fast imaging (five frames/sec) was performed in this channel. To measure residence time of Adf1 in the presence of Cdc8, 2.5 μM of unlabeled Cdc8 was included in the reaction. Single molecules were tracked using MTrackJ . Only siSite of the first tropomyosin Cdc8 binding event on an actin filament was determined by observing TIRFM movies performed at Cdc8 concentrations at the inflection point of the Hill plot (1.25 μM Cdc8). As our resolution limit is 100 nm, and individual Cdc8 molecules may only briefly associate with the actin filament before dissociating or forming a ‘seed’, we cannot determine explicitly the number of Cdc8 molecules in each of these initial association events. At the point of first observation of Cdc8 binding, the length of the actin filament was measured as well as the distance from the pointed end to the site of Cdc8 binding. The barbed and pointed ends of the actin filament were identified by observing photobleaching of the older, pointed end of the actin filament that occurred over time.12 and the reverse process occurring with rate k21 = 0. Once a binding event is observed for a filament, that filament was unable to go back to having been never bound. We described P(t) as a vector representing the populations of the two states.To compute the first binding times of a molecule to a substrate, we modeled the reaction as a master equation with two states, bound and unbound, with unbound transforming to bound at rate kwhere W is a rate matrix whose columns must sum to zero. Hence:grow, the length of the substrate l(t) = vgrowt. With the assumption of uniform binding affinity, the rate of going from unbound to singly bound k12 depended on time, therefore konl(t) = konvgrowt. kon is the rate per unit length of observing Cdc8 stably residing on a filament, which arises from the complex cooperative binding process discussed above and below.In the case of binding to an extending F-actin substrate, if we assumed an average constant growth rate vin which case:P1(τ), times the rate of binding at time τ, k12(τ), hence:Hence, sinceage, P(τage).Given this equation for the binding time, we also determined the probability of binding to a particular site on the actin filament that has been in the filament for an amount of time τgrowt and the probability of Cdc8 binding anywhere along the filament is uniform. The probability of binding at a distance x0 from the end of the actin filament of length l isUnder our assumptions, at time t, the actin filament has length l(t) = vgrow, the probability of binding to a spot of a given age τage isConsequently, given our assumption of a constant average growth rate vage, we integrated To determine the probability that a molecule binds to a part of the substrate of age τon to fit the data in −6 sec−1 nm−1 gave good agreement with the data.where erfc(x) is the so-called complementary error function and was evaluated numerically. Using these equations, we chose kThe validity of the aforementioned expressions was tested by comparison with a simple simulation. The simulation methodology moreover provided a way to mimic the noise level that arose due to sample size limitations. A Monte Carlo scheme was performed by taking discrete time steps of size dt and at each time assuming the probability of binding in that time dt isfrom math import expfrom random import randomdef simulate_binding:   t = 0   while True:     fil_length = v_grow*t     p_bind = 1 - exp(-k_on * v_grow * t * dt)     # pick a random number     #  from zero to one and decide     #  if binding will occur     if random<p_bind:       # choose a random position       #  to bind from barbed end       #  to pointed end       binding_position = random*fil_length       binding_age = binding_position/v_grow       return t,binding_age     t = t + dtdef simulate_experiment:   binding_time_list =    age_list =    for i in range:     t,binding_age = simulate_binding     binding_time_list.append(t)     age_list.append(binding_age)   # histogram/bootstrap age_list   # and binding_time_list   ...An example implementation:http://www.embl.de/eamnet/html/kymograph.html). Spreading rates were determined from kymographs by identifying examples of constant growth over at least 15 s (three frames). The distance of growth over time was calculated to determine a rate of spreading.Elongation rates of individual tropomyosin Cdc8 cables were determined by creating a region of interest (ROI) of an individual actin filament in the actin 488 channel over many time points. The actin ROIs were then applied to the Cdc8 (647) channel and adjusted slightly if necessary to account for movement of the actin filament during channel switching. A kymograph of the Cdc8 647 channel was created from the ROIs using an adapted version of Kymograph - Time Space Plot ImageJ plugin  along the actin filament, we determined the probability of a second Cdc8 cable of length L2 binding across from the first Cdc8 cable. We then compared the probability generated from different models with our experimental results. To account for experimental resolution, we divided the actin filament into a grid of 100 nm segments. In order to compute the probability of the second binding event overlapping with the first Cdc8 cable, we counted the number of places the second cable could bind that overlaps with the first cable of length L1, and divided by the total number of potential places a second Cdc8 cable of length L2 could bind. As a helical actin filament has two grooves, there are twice as many potential binding sites on F-actin unoccupied by Cdc8 as those occupied by a single Cdc8 cable. However, if there is any overlap of second Cdc8 cable binding with the first Cdc8 cable, we assumed that there was only one potential binding face. For random binding, Cdc8 binding to each actin site was given an equivalent likelihood. To account for potential indirect cooperativity, we performed a weighted sum, where the second Cdc8 cable binding is proportionally more or less likely by a factor of c at all sites overlapping with the first cable of Cdc8. The probability of an overlapping binding was therefore given by the following three expressions:To determine whether the proportion of observed second binding events that bind opposite a tropomyosin Cdc8-bound segment  would be2L) was measured, we computed the probability that a cable of length L2 binding would overlap with the first Cdc8 cable (p2). We then re-performed the 37 experiments 5000 times with a chosen indirect cooperativity factor c, choosing randomly with probability p2 whether or not overlapping binding occurred. Binning these events similar to the experimental data, we computed the values shown in We then compared the expected outcomes for random (c = 1X) vs positive or negative indirect cooperativity values (c = 2X or c = 0.5 respectively) . Finallyl of approximately 21.6 nm (assuming an approximate actin spacing of 2.7 nm). The dynamics of Cdc8 molecules associating with this lattice were then simulated using a kinetic Monte Carlo procedure  and a base off-rate koff (1→0). To include the effect of end-to-end cooperativity, the on-rate for unoccupied site ij was multiplied by a factor of w and the off-rate for occupied site ij was divided by a factor of w for each occupied immediate neighbor in the same row  or unoccupied (0) by Cdc8. We described binding site site same row . Using tsame row . In ordesame row . For a lsame row  up to a N = floor(new_time*site_extension_rate)To make kymographs and to compute the occupancy of Cdc8 as accurately as possible, we processed the output raw simulation data in three ways:In TIRFM experiments, ~20% of Cdc8 molecules were labeled. Hence, in our kymographs, we replaced 80% of the occupied sites with a value of 0.The experiments were limited by optical resolution. To approximate this, each remaining occupied site was broadened by filling in adjacent sites in the same row within a radius of 100 nm (±4 sites), the experimental resolution.The two rows were summed together to give fluorescence values of 0, 1, or two at each site along the actin filament.off, we first fixed c, and tried combinations of koff and w that gave (a) the best match to the data shown in on is scanned, and (b) gave kymographs whose first time for observed binding was similar to what was observed experimentally. Due to cooperative binding effects, modeling is required to find a value for kon that is commensurate with experimental data. In off was measured from single molecule Cdc8 events on an actin filament fully coated by Cdc8. In our model, the microscopic koff corresponding to this situation was koffmeasured = koffmodel/w2/c. Using koffmodel values far from those measured in off to the value measured experimentally results in a single value of w that best matched the experimental data , with the results from these simulations shown in off did not alleviate this problem. For a fixed value of c > 1, there is a single w that best matched the experimental data. For c = 1.25, while these simulations matched the experimental data  matched the experimental data  for each experiment. The total actin filament length in the chamber was measured manually by creating ROIs for every actin filament. ROIs for every segment of actin filament present in a bundle were then created, and the ratio of actin filament present in a bundle vs. total actin filament length was quantified.Fold-change in fluorescence intensity over time was taken by taking a total fluorescence measurement of the entire TIRFM field for each frame of the movie. The fold-change in fluorescence of each frame compared to the first frame of the movie was quantified and plotted over time.ROIs were created for all single filaments and two-filament bundles at that time point and total filament length was measured. Severing events that occurred within those filaments prior to the selected frame were quantified, and severing events per micron per second was calculated. We were only able to directly observe severing of single filaments and two-filament bundles, and were unable to determine severing rate within the dense bundles generated by a combination of fimbrin Fim1 and ADF/cofilin Adf1.2, 10 mM ethylene glycol tetraacetic acid [EGTA], and 100 mM imidazole, pH 7.0) and placed in the lower row of the plate. Pyrene fluorescence readout over time was detected using an Infinite M200 Pro  fluorescence plate reader.The spontaneous assembly of 1.5 µM Mg-ATP-actin monomers (10% pyrene-labeled) was carried out in a 96 well plate as described . WT ADF/Fission yeast cells were grown in YE5S media at 25°C for 24–36 hr. Cells were fixed in 100% cold methanol and nuclei (DAPI) and septa  were visualized. For staining, cells were incubated in 300 μL of 50 mM sodium citrate and 4 μL Calcofluor White Stain  at 37°C for 5 min. Cells were then washed with 1 mL of 50 mM sodium citrate, and resuspended in 15 μL sodium citrate and 4 μL DAPI stock . Stained cells were kept on ice until imaging. Cells were imaged using differential interference contrast (DIC) and epifluorescence with an Orca-ER camera  fitted to an IX-81 microscope , with a 60 × 1.4 N.A. Plan Apo objective.Time required for fission yeast strains to assemble a contractile ring was quantified using strains expressing Lifeact-GFP as a general actin marker . Single °C. Fission yeast grown in YE5S were fixed in 16% paraformaldehyde at room temperature for 5 min. Cells were washed with PEM buffer three times at room temperature and permeabilized in PEM with 1% triton X-100 for exactly 1 min. Cells were then spun at 7000 RPM for 30 s and the supernatant was removed quickly. Cells were washed in PEM buffer three times and resuspended in 10 µL PEM buffer following the final wash. Lyophilized phallacidin was resuspended to one unit/µL and 1 µL of resuspended phallacidin was added to 10 µL of cells and incubated at room temperature for 30 min in the dark. Following phallacidin incubation, cells were washed with 1 mL PEM and spun at 7000 RPM for 30 s or until nearly all cells were spun down. Supernatant was removed leaving the resuspended cells in a small volume. Resuspended cells were imaged on glass slides using a Zeiss Axiovert 200M fitted with a 100x, 1.4 NA objective and Yokogawa CSU-10 spinning disk unit  equipped with a 50-milliwatt 473 nm DPS laser and Cascade 512B EM-CCD camera .BODIPY-phallacidin staining of fission yeast to visualize their actin cytoskeleton was performed as described previously . BODIPY-2, 1 mM EGTA, 0.5 mM DTT, 0.2 mM ATP and 90 µM CaCl2 for 1 hr to generate F-actin. F-actin was then incubated with tropomyosin Cdc8 mutants (2 µM) for 20 min at 25°C and spun at 100,000g at 25°C. Supernatant and pellets were separated by 15% SDS-PAGE gel electrophoresis and stained with Coomassie Blue for 30 min, destained for 16 hr and analyzed by densitometry with ImageJ.Sedimentation assays were performed as previously described . 15 μM M In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom, Mohan K Balasubramanian (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Mithilesh Mishra (Reviewer #2); Fred Chang (Reviewer #3).Thank you for submitting your article "Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The editor and referees think that your work on characterizing the competition between tropomyosin, fimbrin, cofilin in actin network organization is exciting.It appears that most of the referee comments can be fixed by rewriting and a better discussion. Please see a consolidated list of referee comments and our expectation during revision in brackets.1) Is the competitive relationship between Fim1 and Cdc8 specific or do other bundling proteins compete with Cdc8 for F-actin binding? The authors have the tools and can easily address potential competition between Ain1 / Fim1/ Cdc8. This becomes particularly important if one considers some of the work of Doug Robinson and colleagues who have investigated micro and nanosecond occupancies of Fimbrin, A-actinin, and cortexilin on actin (Experiment or discussion).2) East et al. (Biochem J) have described tropomyosin mutants that lack the ability for dimer formation. The authors could test use mutant protein as a control to demonstrate Cdc8 cooperativity. This paper was not referred and I hope the authors will rectify this .3) Adf1 preferentially binds to ADP-F-actin and Cdc8 has no preference for the ATP/ADP state of F-actin. How about Fim1? This can be informative on mechanisms of competition to load key binding proteins onto actin filaments. (Experiment or discussion).4). Indirect Cooperativity of Tropomyosin:The authors propose that Tropomyosin engages in both end to end binding and therefore direct cooperative along with an ill-defined indirect cooperativity that facilitates preferential loading of tropomyosin on the opposing actin surface. This is evidenced by an increased Fluorescence intensity of tropomyosin along actin filament. This mechanism of such cooperativity merits some discussion. Tropomyosin mediated actin stabilisation can, in principle, lead to increased probability of tropomyosin loading based on an increased lifetime of a stabilised actin filament. Also the initial loading event whether across or along the filament gives rise to a spike in intensity (taken as the seed of the loading event), that seems to become uniform over time. Does the seed lose its structure? Does it unload after initiating the reaction? Or is it an artefact of photo-bleaching? (discussion).5) The Y-axis reads "Cdc8 pellet normalised to highest value"It's not clear to the reader what highest value is it normalised to. Is it the highest cdc8 pellet recovered or the actin amount. Either case I think it's better to plot cdc8 (pellet) normalised to its associated actin amount. (figure modification).6) Subsection “Tropomyosin Cdc8 is actively displaced from F-actin bundled by fim1.”a) The authors make a strong closing argument in this section –"These findings suggest that the cooperativity of Cdc8 assists not only its assembly onto filaments but also its rapid displacement specifically from F-actin networks bundled by Fim1." However all the evidence strictly points to a fim1 mediated displacement of cdc8 and not any involvement of cdc8's cooperativity in this unloading. I guess that the rapidity of cdc8 stripping lead the authors to this conclusion. Essentially to claim this the authors need to show that fim1 bundling even in a restricted localised zone leads to rapid eviction of the entire tropomyosin filament. Otherwise and as it stands currently, fim1 coats the entire actin filament along its length and consequently cdc8 is excluded. The fast dynamics can be attributed to rapid actin bundling (fim1 association) and physical eviction of tropomyosin without invoking tropomyosin cooperativity. (perform an experiment or tone down the conclusions).b) The authors, although not explicitly, seem to wrongly indicate the contractile ring is devoid of cofillin. (clarify this point). The editor and referees think that your work on characterizing the competition between tropomyosin, fimbrin, cofilin in actin network organization is exciting.It appears that most of the referee comments can be fixed by rewriting and a better discussion. Please see a consolidated list of referee comments and our expectation during revision in brackets.1) Is the competitive relationship between Fim1 and Cdc8 specific or do other bundling proteins compete with Cdc8 for F-actin binding? The authors have the tools and can easily address potential competition between Ain1 / Fim1/ Cdc8. This becomes particularly important if one considers some of the work of Doug Robinson and colleagues who have investigated micro and nanosecond occupancies of Fimbrin, A-actinin, and cortexilin on actin (Experiment or discussion).eLife as an accompanying 'Research Advance' article that is tentatively entitled "Cooperation between Tropomyosin Cdc8 and α-actinin Ain1 prevents fimbrin Fim1 association with contractile ring F-actin." In brief, we have discovered that the competitive relationship is specific to Fim1 and Cdc8.We agree that this is an extremely interesting and important question, which is a major area of investigation that we have been actively pursuing. In fact, we are preparing a new manuscript describing this work that we will soon be submitting to Conversely,  Ain1 andWe also agree that it is important to reference work from the Robinson lab and the importance of dynamics in mediating ABP sorting, and have added a new section to the Discussion: “We suspect that Fim1’s long residence time on F-actin bundles enhance its ability to create regions of high Fim1 occupancy that displace Cdc8, suggesting that ABP dynamics (on-/off-rate) may partially dictate the ability of an ABP to compete. An ABP’s dynamics could be driven by its intrinsic biochemical properties , post- translational modifications , or mechanical stresses within the F-actin network , resulting in the fine-tuning of ABP sorting by many factors.”2) East et al. (Biochem J) have described tropomyosin mutants that lack the ability for dimer formation. The authors could test use mutant protein as a control to demonstrate Cdc8 cooperativity. This paper was not referred and I hope the authors will rectify this .The mutants referred to in this paper are still capable of forming Cdc8 dimers, but have mutations in the end-to-end binding regions that inhibit or enhance the ability of Cdc8 to polymerize into cables. Due to the unusual binding dynamics of Cdc8 on F-actin, it is difficult to assess our model of Cdc8 indirect cooperativity using these mutants. An ideal experiment would enhance the ability of Cdc8 to bind to F-actin while inhibiting its end-to-end binding. However, a mutant that is able to strongly associate with actin without being able to polymerize has not been identified. Therefore, though these mutants are certainly interesting, it would be difficult to address our model of indirect cooperativity with use of these mutants, as they only inhibit or enhance ability of Cdc8 to polymerize. We agree that this is an important paper to refer to and have included the following new statement: “Mutations in the N- or C-terminal domains of Cdc8 affect its ability to bind cooperatively and polymerize on an actin filament .”3) Adf1 preferentially binds to ADP-F-actin and Cdc8 has no preference for the ATP/ADP state of F-actin. How about Fim1? This can be informative on mechanisms of competition to load key binding proteins onto actin filaments. (Experiment or discussion).Our lab has previously determined that fimbrin Fim1 has no preference for the ATP/ADP state of F-actin . We agree that the mechanisms of initial loading of key ABPs is particularly interesting and is the subject of current research in our lab.4) Indirect Cooperativity of Tropomyosin:The authors propose that Tropomyosin engages in both end to end binding and therefore direct cooperative along with an ill-defined indirect cooperativity that facilitates preferential loading of tropomyosin on the opposing actin surface. This is evidenced by an increased Fluorescence intensity of tropomyosin along actin filament. This mechanism of such cooperativity merits some discussion. Tropomyosin mediated actin stabilisation can, in principle, lead to increased probability of tropomyosin loading based on an increased lifetime of a stabilised actin filament. Also the initial loading event whether across or along the filament gives rise to a spike in intensity (taken as the seed of the loading event), that seems to become uniform over time. Does the seed lose its structure? Does it unload after initiating the reaction? Or is it an artefact of photo-bleaching? (discussion).We agree that the mechanism of indirect cooperativity merits further discussion, and have also thought that this may be a result of stabilization of the actin filament, particularly as it is known that Cdc8 enhances the persistence length of F-actin. We have added new additional discussion:“Tropomyosin is known to increase the persistence length of F-actin . As a result, the stabilizing effect of an initial Cdc8 binding to one side of the actin filament may favor binding of a second Cdc8 to the opposing side. Additionally, if the rigidity provided by initial Cdc8 binding is propagated slightly further up or down the actin filament, more tropomyosin molecules could associate with the actin filament as a result of an increase in F-actin rigidity.”The uniformity over time in fluorescence appears to be an artifact of photobleaching, as it happens faster when we image more frequently. However, we cannot rule out the possibility that tropomyosin molecules are leaving the cable, potentially at ‘gap’ regions. The longer cables appear to remain intact, though we do observe small seeds  that do grow and shrink more frequently.5) The I76C and D142C Cdc8 mutants are both suitable for TIRF microscopy experiments, and in fact, we use both in the lab. However, we have found that I76C has been easier to label well consistently, for reasons that are unclear. Additionally, our updated analysis both in vitro and in vivo  and 2 shb) The Y-axis reads "Cdc8 pellet normalised to highest value"It's not clear to the reader what highest value is it normalised to. Is it the highest cdc8 pellet recovered or the actin amount. Either case I think it's better to plot cdc8 (pellet) normalised to its associated actin amount. (figure modification).These plots were normalized to the highest Cdc8 recovered. When we plot Cdc8 in the pellet normalized to its associated actin, all mutants appear to bind F-actin similarly. We have changed the graph to show Cdc8 pellet normalized to associated actin .6) Subsection “Tropomyosin Cdc8 is actively displaced from F-actin bundled by fim1.”a) The authors make a strong closing argument in this section –"These findings suggest that the cooperativity of Cdc8 assists not only its assembly onto filaments but also its rapid displacement specifically from F-actin networks bundled by Fim1." However all the evidence strictly points to a fim1 mediated displacement of cdc8 and not any involvement of cdc8's cooperativity in this unloading. I guess that the rapidity of cdc8 stripping lead the authors to this conclusion. Essentially to claim this the authors need to show that fim1 bundling even in a restricted localised zone leads to rapid eviction of the entire tropomyosin filament. Otherwise and as it stands currently, fim1 coats the entire actin filament along its length and consequently cdc8 is excluded. The fast dynamics can be attributed to rapid actin bundling (fim1 association) and physical eviction of tropomyosin without invoking tropomyosin cooperativity. (perform an experiment or tone down the conclusions)We agree that Cdc8 displacement appears to be directly mediated by Fim1, but also posit that Cdc8 cooperativity may enhance its Fim1-mediated removal, resulting in an ‘all-or-nothing’ removal of Cdc8 cables from the actin filament across short ranges. The stripping of Cdc8 cables from initial sites of removal is what led us to this conclusion, but we agree that we have overstated the claim. We have toned down our conclusions and it now reads “These findings demonstrate that Fim1-mediated bundling displaces Cdc8 from the actin network. Additionally, as Cdc8 appears to be rapidly ‘stripped’ from a few initial dissociation points, it is possible that the cooperativity of Cdc8 assists not only its assembly onto filaments but also its rapid displacement specifically from F-actin networks bundled by Fim1.”b) The authors, although not explicitly, seem to wrongly indicate the contractile ring is devoid of cofillin. (clarify this point).We agree that the wording is confusing and have clarified the point to now read “Though Cdc8's presence at the contractile ring and actin cables likely prevents the unwanted association of several types of ABPs at those F-actin networks, opposing mechanisms must be in place to prevent Cdc8 from associating with actin patches“.


Opuntia streptacantha cladodes. Cladodes segments were exposed to salt stress at 0, 100, 200, and 300 mM NaCl for 8, 16, and 24 h. The results showed that salt stress reduced chlorophyll content, Fv/Fm, ΦPSII, and qP values. Under the highest salt stress treatments, the chloroplasts were densely clumped toward the cell center and thylakoid membranes were notably affected. We analyzed the effect of exogenous catalase in salt-stressed cladode segments during 8, 16, and 24 h. The catalase application to salt-stressed cladodes counteracted the NaCl adverse effects, increasing the chlorophyll fluorescence parameters, photosynthetic-pigments, and avoided chloroplast clustering. Our results indicate that salt stress triggered the chloroplast clumping and affected the photosynthesis in O. streptacantha chlorenchyma cells. The exogenous catalase reverted the H2O2 accumulation and clustering of chloroplast, which led to an improvement of the photosynthetic efficiency. These data suggest that H2O2 detoxification by catalase is important to protect the chloroplast, thus conserving the photosynthetic activity in O. streptacantha under stress.In arid and semiarid regions, low precipitation rates lead to soil salinity problems, which may limit plant establishment, growth, and survival. Herein, we investigated the NaCl stress effect on chlorophyll fluorescence, photosynthetic-pigments, movement and chloroplasts ultrastructure in chlorenchyma cells of  Previous studies have shown that organelles, such as mitochondria, peroxisome, and chloroplast are constantly moving within the cells taking specific positions to maximize their metabolic activities during changing environmental conditions4. In particular, the chloroplast movement in response to light (photo-relocation) is essential for the survival of plants under extreme light conditions6. Additionally, chloroplast movement has a protective role in response to other abiotic stresses, such as drought and low temperatures9. Chloroplast movement has been extensively studied in the model plant Arabidopsis thaliana, operating with C3 type photosynthesis5. However, only a few studies have been undertaken about chloroplast movement in C4 and CAM (Crassulacean Acid Metabolism) plants11. Previously, our research group reported that the Opuntia streptacantha chloroplasts were grouped together within the cells under combined light and water stress, which probably maintain its photosynthetic process active in CAM plants9.To ensure their survival, plants have evolved to optimize the capture of energy and nutrients12. The water loss is minimized because during the daytime CAM plants photosynthesize with closed stomata, using the CO2 that was stored in the vacuole in form of organic acids, mainly malic acid, during night12. Thus, CAM plants are able to survive under extreme abiotic stress conditions, predominantly scarcity of water and extreme temperatures, in contrast to C3 and C4 plants13.Particularly, the high plasticity in the CAM photosynthetic pathway is one of the most successful physiological strategies for plant acclimation and adaptation to water shortageOpuntia streptacantha is an endemic cactus from Mexico, which is distributed along the southern Chihuahuan Desert14. It performs a CAM type photosynthesis, which permits a successful establishment of intracellular levels of the plant in arid and semiarid regions of many countries15. Despite the ability of CAM plants to overcome water stress, not all CAM plants are successful to cope salinity12.16. An early effect of salt stress is stomatal closure, which leads to a restriction of CO2 diffusion into the cells and chloroplasts17. Subsequently, the increase of reactive oxygen species (ROS) levels, can seriously affect the plant’s photosynthetic machinery19. However, ROS are also important signaling molecules involved in the control of plant growth, development, photosynthetic functions, and responses to biotic and abiotic stress21. In particular, the hydrogen peroxide (H2O2) is the most significant non-radical ROS22. H2O2 is produced predominantly during the photosynthesis and photorespiration process in the apoplast, chloroplast, peroxisome, and mitochondria23. Plants possess efficient scavenging systems for ROS, which protect them from destructive oxidative reactions. The principal H2O2 scavenging enzyme in plants is catalase (CAT), which directly converts H2O2 into H2O and O224. Likewise, ascorbate peroxidase (APX) and glutathione peroxidase (GPX) are important enzymes essentials for the elimination of H2O2 levels in the cell25. The balance between ROS generation and ROS-scavenging during exposure to stressful environments is essential to regulate the mechanisms of ROS signaling in plants27.It is known that salinity affects the photosynthesis process in plants2 uptake in Opuntia plants under salt stress have been previously reported30; however, little information about other physiological data related to salinity has been provided. In this study, our main objective was to examine the effect of NaCl treatments on chloroplast movement in chlorenchyma cells of O. streptacantha. Through optical microscopy, we observed the chloroplast arrangement under different treatments with NaCl . Additionally, we measured diverse physiological parameters, such as the chlorophyll fluorescence, photosynthetic pigments content, and ultrastructure of chloroplasts to determine the effect caused by NaCl treatments. We found that the H2O2 accumulation, clustering of the chloroplasts, and a decline in photosynthetic activity were consequences of salinity in O. streptacantha. Finally, exogenous application of CAT enzyme was used to counteract the effects produced by salt treatment. Our data suggest that H2O2 accumulation is relevant for chloroplast clustering under salinity in O. streptacantha.The negative effects on growth and COO. streptacantha to evaluate the effect of salinity on photosynthesis, by using cladode segments exposed to salt treatments at 0, 100, 200, and 300 mM NaCl for 8, 16, and 24 h. The values of the maximum quantum yield of photosystem II (Fv/Fm), the effective photochemical quantum yield of PSII (ΦPSII), and the photochemical quenching (qP) showed a decreasing trend related to NaCl concentration and treatment duration  and the non-photochemical fluorescence quenching (NPQ) values were significantly increased  was significantly reduced at 200 and 300 mM NaCl compared to the control , and carotenoids (x + c) content in cladodes segments exposed to salt treatments at 0, 100, 200, and 300 mM NaCl during 8, 16, and 24 h. We observed that the chlorophyll a, and total (a + b) contents tends to decline along with the exposure time to NaCl concentrations  levels at 100 and 200 mM NaCl concentrations compared to the control. However, no changes were observed in the Chl a, and total (a + b) levels at 16 h under the salt treatments evaluated. After 24 h of the salt treatments, we found a decrement of chlorophyll a, and total (a + b) levels in all the salt concentrations tested  at 8 h of salt treatments. Meanwhile, the Chl a/b ratio was significantly reduced at 16 and 24 h for all the salt treatments /(x + c) was significantly reduced only at 24 h of salt treatments . At the ultra-structural level, chloroplast distortion was observed in the cladode segments exposed to salt treatment for 8, 16, and 24 h compared to the control . In this regard, our data indicate that the application of CAT may inhibit the clustering of chloroplasts during salt stress in O. streptacantha chlorenchyma cells.To determine if the exogenous catalase (CAT) application may counteract the aggregative effect of chloroplasts under salt stress, we incubated cladode segments in solutions with 200 mM NaCl supplemented with 100, 200, and 300 UmL8 h Fig. . Under t2O2 scavenging activity under salt treatment, we monitored the H2O2 using the fluorescent signals of specific fluorescent probes (DCF-DA) on O. streptacantha cladode cells exposed to 200 mM NaCl + 300 UmL−1 CAT during 8 h , and NPQ remained close to the control without salt stress  got photochemically inactive, which reduced electron transport capacity in PSII and that decreased the photosynthetic capacity of O. streptacantha cladodes under salt stress.In this study, we analyzed the effect of salt stress on the movement of chloroplasts in the chlorenchyma cells of O. streptacantha cladodes was analyzed. Our data showed that the decrease in the content of chlorophylls at 8 and 24 h was more pronounced at a low than at a higher salt concentration. One possible explanation could be that O. streptacantha cells, when sensing high salt concentrations activate several mechanisms to induce de novo chlorophyll synthesis, and thus counteract osmotic and ionic shock. This behavior has been previously described in cell lines of Medicago sativa and Nicotiana tabacum, which showed a greater accumulation of chlorophylls under salt stress33. These authors postulate that these changes may be an indicative of physiological processes activation in chloroplasts against saline stress. Therefore, O. streptacantha cells could activate mechanisms such as chloroplast biogenesis, osmoprotectants synthesis pathways, detoxification, heat shock protein and late embryogenesis abundant proteins involved in chloroplast protection. Moreover, no significant changes in the chlorophyll content at 16 h of NaCl treatments was observed, which could be a feature of acclimation by the re-establishing cellular ion homeostasis in cladode segments under salt stress.In addition, the effect of NaCl treatments on the photosynthetic pigment content in chlorenchyma cells of 34. The reduction in the chlorophyll a/b ratio in cladode segments under the salt treatments showed in O. streptacantha can be interpreted as an enlargement in the light-harvesting complex II (LHCII) antenna of PSII. Thus, we suggest that the increase in antenna size could reduce the excitation energy from the antenna to RCs of PSII, or as a direct response of photosynthetic apparatus to salt stress. Similar results were presented by Demetriou et al.35 who reported that salt stress triggered an increase in the photosynthetic effective antenna size in Scenedesmus obliquus under low light conditions.We showed that salt stress causes photosynthesis damage, which in turn would provoke the clustering of chloroplasts like a mechanism of protection to prevent light to penetrate to deeper layers and then reduce the photo-oxidation. However, plants have additional mechanisms to protect themselves against damage by an excess of energy36. The ultrastructural analysis in O. streptacantha chloroplast exhibited that salt treatment induced thylakoid membranes stacking at 8 and 16 h. Dynamics in the stacking of the thylakoid membranes are essential for regulatory processes of the photosynthesis under different abiotic stress conditions34. The ability to control the lateral separation of PSI from PSII is considered a functional consequence of grana stacking to minimize the formation of ROS in the PSI through of the decrease of electron transport between photosystems38. Thus, our results suggest that changes of thylakoid membranes permit a balance in the excitation energy between the two photosystems37. These features can therefore be considered as a response of O. streptacantha cladodes to salt stress.Moreover, it has been demonstrated that a re-arrangement on antenna leads to the stacking of thylakoid membranes43.Instead, the ratio of chlorophyll to carotenoids at 24 h for the NaCl treatments was low, which indicate damage to the photosynthetic machinery. This result was consistent with the breakdown of chlorophyll caused by chloroplast damage with 200 mM NaCl at 24 h. Several studies have reported that salt stress induce the degradation of photosynthetic pigments in plants by the accumulation of toxic ions and ROS, changing the ultrastructure of the photosynthetic apparatus1-qP) on the photosynthetic machinery in O. streptacantha cladodes. The aggregative arrangement of chloroplasts in O. streptacantha seedlings also occurs under drought and high sunlight conditions9. Our research group has proposed that chloroplasts move towards the vacuole facilitating malate transportation; thus maintaining the photosynthetic activity under water deficit. The chloroplast-clumping phenomenon is a typical mechanism that has been observed in plants as a response to high solar radiation11, salinity39, drought44, and low temperatures8. It has been proposed that the aggregation of chloroplasts may provide protection against photodamage and help to maintain the photosynthetic activity under stressful conditions45. Therefore, this phenomenon could be a common adaptive strategy used by plants for their survival under abiotic stress.Although the chloroplasts could experience damage under salt stress, our results suggest that the clumping of chloroplasts plays an important role to minimize the excitation pressure 47, glutathione (GSH)49, and ∝-tocopherols50. It is known that enzymatic antioxidants such as CAT provides a very energy-efficient mechanism to remove H2O251. A high level of endogenous CAT is essential to maintain the antioxidant system that protects plants from oxidative damage due to various environmental stresses52. Thus, the exogenous CAT applied to cladode segments of O. streptacantha produced a protection against oxidative damage by H2O2 scavenging activity.We also examined the effect of exogenous catalase (CAT) on chloroplasts clumping of 2O2 production in acclimation response of plants to salinity54. It has been proposed that H2O2 accumulation in the apoplast could activate a signal for the chloroplast due to their location close to the plasma membrane. Then, the chloroplast may transmit the ROS signaling to the nucleus for the photosynthesis acclimation through nuclear gene expression20. Moreover, Wen and Zhang55 reported that high blue light exposure can induce H2O2 generation in the plasma membrane, and that H2O2 is involved in chloroplast movements in Arabidopsis thaliana. Thus, we proposed that H2O2 generated by salt stress could be acting as a signaling molecule which promotes clumping of chloroplasts, particularly as an acclimation mechanism for mitigation of photo-inhibition in salt-stressed cladodes of O. streptacantha. However, the role of H2O2 in chloroplast movement is still poorly understood.The apoplast is an important site for H2+ ([Ca2+]cyt), are largely recognized to be important signaling messengers of many biological responses56. It is known that high NaCl concentrations, particularly under excess of chloride (Cl−) ions in the cytoplasm, leads to an increase of [Ca2+]cyt concentrations, which initiates the stress signal transduction pathways in plants under salt stress59. Furthermore, studies in Arabidopsis leaves reported that calcium is involved in the signal transduction for the movement of chloroplast in response to blue light62. The authors work shows that the stress by high blue light can increase [Ca2+]cyt, which may trigger the activity of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) to generate H2O2. In turn; the H2O2 generated may promote the chloroplast movements. Thus, it is possible that the clustering of chloroplast in cladodes segments can be regulated by internal Ca2+ stores, produced by salt stress. However, the cross-talk between Ca2+ and H2O2 in the regulation of chloroplast movements in O. streptacantha cells is an open question arising from this work that demands future research efforts. Finally, the physiological responses of CAM cacti to salinity are complex and the mechanisms underlying this phenomenon have not been completely elucidated, opening a variety of exciting new questions.In higher plants, ROS and cytosolic Ca2O2 in the O. streptacantha cells. We suggest that H2O2 acts as a messenger molecule for the clustering of chloroplasts. The exogenous application of CAT alleviates salt-induced oxidative stress in O. streptacantha cladodes most likely through H2O2 scavenging activity. CAT activity avoids chloroplasts clustering and protects photosynthetic machinery function in salt-stressed O. streptacantha. In this regard, further studies about the effect of H2O2 in chloroplast clustering will help to understand the role of this phenomenon during stress.The chloroplasts are usually more sensitive to salinity than other organelles. Our findings show that salt stress causes photosynthesis damage and accumulation of HOpuntia streptacantha seeds were collected in 2007 by Delgado-Sánchez et al.9 from Mexquitic de Carmona municipality San Luis Potosi, Mexico, . Seeds were sowed in LM-1 substrate  and watered daily until germination. After one month, the seedlings were transplanted in pots containing peat moss substrate . Pots were maintained under greenhouse conditions at 1059 ± 5.5 μmol m−2 s−1 photosynthetic photon flux density (PPFD) with 23 °C average air temperature, and watered every other day, during six months. Then, the plants were transferred to a growth chamber for one month at 25 °C under 16 h light (300 μmol m−2 s−1 PPFD) and 8 h dark cycle.O. streptacantha plants. For salt treatments, the cladode segments were completely immersed in NaCl solutions  or on distilled water as control for 8, 16, and 24 h. NaCl concentrations were chosen based on previous studies performed in Opuntia species64. The assays were performed using three different cladodes of O. streptacantha plants  and we take three segments of cladode per plant  for each treatment, under continuous white light (300 μmol m−2 s−1) at 25 °C.Segments of cladode (5 × 8 × 0.3 mm) were hand-cut in transverse sections from −1 respectively, were added to 200 mM NaCl solution. Subsequently, the cladode segments were completely immersed in these mixtures (NaCl + CAT) for 8, 16, and 24 h. As control, we used distilled water without NaCl or CAT. The CAT concentration was selected according to Aroca65. The assays were performed using three different cladodes of O. streptacantha plants  and we take three segments of cladode per plant  for each treatment, under continuous white light (300 μmol m−2 s−1) at 25 °C.For CAT mixed with NaCl solution treatments, the CAT stock solution was prepared by pre-dissolving 1 mg of CAT (Sigma-Aldrich Ref: C1345) in 1 mL 50 mM potassium phosphate buffer pH 7.0. The CAT solutions at final concentrations of 100, 200, and 300 UmL66 and can also be applied to know how plants respond to abiotic stress factors67. Cladode segments of 10 mm diameter were removed from O. streptacantha plants using forceps and scalp. Subsequently, these were subjected to the aforementioned treatments. Chlorophyll fluorescence was measured using a MINI-PAM II fluorometer  following the manufacturer’s instructions. The chlorophyll fluorescence measurements were realized using a pulse the actinic light with an intensity of 820 μmol m−2 s−1. The maximum quantum yield of photosystem II (Fv/Fm) was determined after dark adaptation of cladodes segments for 30 min. The Fv/Fm values were calculated as described by Kitajima and Butler68. On the other hand, light-adapted cladodes segments were used to measure the fluorescence parameters as follows: the effective photochemical quantum yield of PSII was calculated using the equation: ΦPSII = (Fm′ − F)/Fm′ = ΔF/Fm′ presented by Genty et al.69. The photochemical quenching was calculated as qP = (Fm′ − F)/(Fm′ − FO′) and used to determine the fraction of closed (reduced) PSII reaction centers, also known as excitation pressure, and calculated as 1–qP. The non-photochemical fluorescence quenching, NPQ = (Fm − Fm′)/Fm′ was determined according to Bilger and Björkman70. The photosynthetic electron transport rate (ETR) was estimated with the following equation ETR = ΦPSII × PPFD × 0.5 × 0.84, where PPFD is photosynthetic photon flux density, the factor 0.5 assumes that photosystems II and I are similarly excited by the irradiance. The factor 0.84 considers that only 84% incident irradiance will be absorbed by the two photosystems71. The assays were performed using three different cladodes of O. streptacantha plants  and we take three segments of cladode per plant  for each treatment (n = 9).The chlorophyll fluorescence analysis is a powerful technique that allows to obtain detailed information about of the process of photosynthesis in plantsa and b) and carotenoids extraction was performed according to the methodology reported by Lichtenthaler73. The cladode chlorenchyma fresh segments were homogenized in 80% acetone and incubated in dark at 4 °C for 5 min. Subsequently, it was centrifuged at 13,000 rpm at 4 °C for 5 min. The chlorophyll and carotenoids contents were measured in 200 μL supernatant using a microplate reader  at 663 (chlorophyll a), 646 (chlorophyll b), and 470 (carotenoids) nm wavelengths. The assays were performed using three different cladodes of O. streptacantha plants  and we take three segments of cladode per plant  for each treatment (n = 9). The pigments content, the ratio of chlorophyll a (Chl a) to chlorophyll b (Chl b) (Chl a/b), and Chl a and Chl b to total carotenoids (a + b)/(x + c) ratio were estimated using the equations proposed by Lichtenthaler73:The chlorophyll  subjected to the treatments described below were observed without fixation under a light microscope Leica DM2000 . The photographs were obtained and digitized with LAS Imaging Software (Leica). For the transmission electron microscope (TEM), the segments of cladodes were treated with a fixative solution  during overnight at 4 °C. Then, they were washed using Sorensen’s phosphate buffer and dehydrated with ethanol; subsequently, the samples were polymerized in a fresh resin at 60 °C as described by Cocoletzi et al.74. Ultrathin sections were contrasted using aqueous uranyl acetate (2% w/v) and aqueous lead citrate (2% w/v). Samples were examined with TEM JEOL 200CX  using a 100 kV acceleration voltage.To analyze the chloroplast arrangement on et al.38. The distances of stacking repeat unit (R), which includes the two thylakoid membrane bilayers (M) and the widths of one partition gap (P) were realized with Image J software75. Subsequent, the thickness of the thylakoid lumen (L) were estimated using the equations proposed by Kirchhoff et al.38: L = R − M − P. The measurements of lumen thickness were made on grana lamellae (GL) using three separate micrographs and we take three grana lamellae per micrographs for each treatment (n = 9).The ultrastructural analysis of chloroplasts to determine the thickness of the thylakoid lumen was realized using the methodology reported by Kirchhoff 2O2 signals, segments of cladode (5 × 8 × 0.3 mm) subjected to the treatments described below were transferred to 25 μM 2′, 7′–dichlorofluorescein diacetate (DCF-DA) (Sigma-Aldrich Ref: C1345) dissolved in 10 mM Tris-HCl, 50 mM KCl (pH 7.2) and incubated in dark for 30 min at 30 °C. Subsequent, the segments were washed twice with 10 mM Tris-HCl, 50 mM KCl (pH 6.1) for 30 min, images were visualized using an Epi-fluorescence microscope . The assays were performed using three different cladodes of O. streptacantha plants  and we take three segments of cladode per plant  for each treatment (n = 9).For observer H76, using Duncan’s multiple range test at the P < 0.05 level of significance between treatments and time (hours). The results are expressed as mean values ± SD (standard deviation) (n = 9).All data obtained from chlorophyll fluorescence parameters and pigments content were statistically analyzed with STATISTICA version 7 softwareSupplementary Information


Systemic lupus erythematous (SLE) is a complex autoimmune disease with female predominance, particularly affecting those of childbearing age. We performed analysis of three genome-wide genotyping datasets of populations of both Chinese and European origin.This study involved 5695 cases and 10,357 controls in the discovery stage. The lead signal on chromosome X was followed by replication in three additional Asian cohorts, with 2300 cases and 4244 controls in total. Conditional analysis of the known associated loci on chromosome X was also performed to further explore independent signals.GPR173 was found to be significantly associated with SLE , whereas conditional analysis provided evidence of a potential independent signal in the L1CAM-IRAK1-MECP2 region in Asian populations (rs5987175 [LCA10]).Single-nucleotide polymorphism rs13440883 in We identified a novel SLE susceptibility locus on the X chromosome. This finding emphasizes the importance of the X chromosome in disease pathogenesis and highlights the role of sex chromosomes in the female bias of SLE.The online version of this article (10.1186/s13075-018-1590-3) contains supplementary material, which is available to authorized users. Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder that leads to autoantibody production and multiorgan damage. Both genetic and environmental components are known to contribute to the disease. SLE demonstrates a dramatic sex bias and predominantly affects women of childbearing age, with an approximately 9:1 female-to-male ratio.Sex hormones probably play a vital role in the female predominance of the disease. In addition, premenstrual and postmenopausal women show a higher prevalence of SLE than men of an identical age group , 2, sugg+ T cells was found to be defective in patients with SLE, and several SLE-associated X chromosomal genes and microRNAs were found to be overexpressed in T cells of female patients with SLE ), 460 cases and 965 controls from Thailand (Thailand replication panel [TH_REP]), and 1102 cases and 2327 controls from Anhui (Anhui replication panel [AH_REP]) (Table Pmeta < 1 × 10− 3 and low LD with the reported SNP (r2 < 0.5) were selected for further examination, and the SNPs with a conditional P value less than 0.01 were considered as potential independent signals.All of the known SLE risk loci in the X chromosome identified in previous association studies, as well as the SNPs in the surrounding regions (± 200 kb), were closely examined. Pairwise linkage disequilibrium (LD) among the SNPs was calculated to detect any potential independent signals. SNPs with association L1CAM- MECP2 region were much stronger than the others, the analysis was different for this region. Eighty-eight SNPs with genome-wide significance (P < 5 × 10− 8) were selected, instead of using the P < 1 × 10− 3 threshold used in the other regions. An LD block was plotted, and the top SNPs in each block were selected for a conditional analysis to test for independent associations. Only the SNPs with a conditional P value less than 0.01 were considered as potentially independent.Because the association signals in the . [. [i (ni) is multiplied by the natural logarithm of the corresponding OR, which is then summed and divided by k  to obtain the average genetic risk score (aGRS):In order to measure the disease risk for SLE for each individual and compare it between the sexes and populations, we performed a modified calculation of genetic risk scores described by Hughes et al. . Based o. [. , a genett test.A total of 1659 Asian SLE samples (Hong Kong and Anhui combined) and a total of 4036 European SLE samples were included to calculate the aGRS, and the differences between male and female cases were analyzed using Student’s . [We used the intragenomic replicates (IGR) method described by Cowper-Sal lari et al.  to predi.  website,Our study consisted of three sets of GWAS data, including two GWASs from China , 14 and Pmeta < 1 × 10− 4 level of significance were further analyzed  excluded 39 SNPs from the list, leaving 9 SNPs with potential independent signals .On the basis of our meta-analysis results, after removing known associated regions as mentioned above, 48 SNPs with Pmeta value of 7.53 × 10− 9, reaching genome-wide significance  showed a final Pmeta < 1 × 10− 3). However, after conditional analysis, none of them remained significant. Thus, based on the samples available, rs13440883 is the only independent association signal confirmed in this region.Conditional analyses were performed on Asian and European GWAS data to test for potential independent signals in this locus. Within the ± 200-kb window, there were 60 SNPs with association signals (r2 > 0.8) with rs13440883 in both European and Asian populations and different TFs with binding peaks overlapping the corresponding SNPs are highlighted in Additional file r2EUR = 0.9384; r2Asian = 0.9915), was a TF binding hot spot in the coding region of GPR173.Data on histone modification, DNase hypersensitivity, and TF binding, accessible using ENCODE , were usP = 3.54 × 10− 5) in the chromatin-binding intensity of SMARCA4 for the alternative allele. For rs11091720, the prediction showed a significant decrease in the intensity of L3MBTL2 (P = 2.31 × 10− 13) and CTCFL (P = 3.67 × 10− 10), as well as an increase in the intensity of CTCF (P = 6.22 × 10− 11) and JUN (P = 0.0064) for the alternative allele. SMARCA4 is a component of a large ATP-dependent chromatin-remodeling complex (SNF/SWI) that is required for transcriptional activation of genes normally repressed by chromatin. CTCF forms methylation-sensitive insulators that may regulate X chromosome inactivation. The function of L3MBTL2, CTCFL, and JUN was not previously known. However, before experimental validation, we had to treat the results of this in silico analysis carefully, which is discussed later.The IntraGenomic Replicates tool  was usedPRPS2, LINCO1420, CXorf21, and IRAK1. The susceptibility gene TLR7 is not examined in this analysis due to poor coverage of this region. All the SNPs within a ± 200-kb window centered on the reported SNPs were filtered by association P values and pairwise LD (r2) with each other  attained genome-wide significance (P < 5 × 10− 8)  in LCA10 exhibited an independent contribution toward SLE susceptibility. After adjusting for the effect of the known independent SNPs  reported in our previous study [Meta-analysis of the GWAS datasets showed 88 SNPs in the Pmeta = 7.53 × 10− 9, ORmeta = 1.16) within GPR173, as well as a potential independent signal  within LCA10 of the L1CAM-MECP2 region on chromosome X as being associated with SLE.In this cross-population meta-analysis of three GWAS datasets and further replication in three additional cohorts, with a total of 7995 cases and 14,601 healthy controls, we successfully identified a novel variant . SMARCA4 is involved in the glucocorticoid receptor regulatory network, which was reported to affect sex differences in the prevalence of inflammatory disease [The novel risk-associated variant, rs13440883, is located within the intron between the second and third exons of e ENCODE  project tes tool  predicte disease . Howeverr2 > 0.8) with rs13440883 detected in the present study, rs11091720 is the only one located in the coding region of GPR173 (synonymous). It is also the only one with multiple TF binding data  173, a member of the G protein-coupled receptor 1 family. In our previous study, an SNP in the upstream of GPR19 was identified as an SLE susceptibility [Although there is as yet no existing evidence showing their influence on the gene’s expression, both rs13440883 and rs11091720 are located within the tibility –32.L1CAM-MECP2 region, with 88 SNPs reaching genome-wide significance in the discovery panel alone. A conditional test using logistic regression was performed to test for independent signals, and rs5987175 in LCA10 was found to remain significant in Asians, after adjusting for the effect of three known independent SNPs   was higher than in Asians . Thus, a larger sample size would be needed to acquire adequate power to assess independent signals in this region in Europeans.Consistent with previous studies on the X chromosome, the strongest association signal in our meta-analysis was from the The novel association signal detected in the present study has a larger effect size in males than in females Fig. , which iGPR173 and a potential independent signal (rs5987175) in LCA10. Further functional annotation and in silico analysis provided plausible candidates for functional causal variants, although experiments are still needed to validate the prediction. A larger sample size is needed to further confirm the independence of rs5987175 in Europeans. The fact that males have a higher effect size than females indicates that the currently identified X-linked SNP is probably not contributing to the female prevalence of the disease. Further investigations are still needed to elucidate the mechanism of the sex bias of SLE.In our present cross-population meta-analysis following replication, we identified a novel SLE-associated variant (rs13440883) in Additional file 1:Table S1. Complete list of ChIP-seq files used in Intragenomic Replicates (IGR) analysis. Table S2. List of the 48 SNPs with association P value smaller than 1 × 10-4. Table S3. List of the candidate X-linked SLE susceptibility genes. Table S4. List of the 88 SNPs surpassing genome wide significance in L1CAM-MECP2 region. Table S5. Conditional logistic regression results in both Asian GWAS and European GWAS. Table S6. The list of SNVs used in Figure S3A. Figure S1. QQ plot for the cross-population X chromosome meta-analysis data. Figure S2. The LocusZoom Plot showing association significance and local LD for the region around rs13440883 (±200kb). Figure S3. Identification of functional risk-associated SNV shared between Europeans and Asians. Figure S4. LD patterns of the risk-associated SNPs in L1CAM-MECP2 region. (PDF 2421 kb)


Amyloid-β (Aβ) and tau pathologies are intertwined in Alzheimer’s disease, and various immunotherapies targeting these hallmarks are in clinical trials. To determine if tau pathology influences Aβ burden and to assess prophylactic benefits, 3xTg and wild-type mice received tau immunization from 2–6 months of age. The mice developed a high IgG titer that was maintained at 22 months of age. Pronounced tau and Aβ pathologies were primarily detected in the subiculum/CA1 region, which was therefore the focus of analysis. The therapy reduced histopathological tau aggregates by 70–74% overall , compared to 3xTg controls. Likewise, western blot analysis revealed a 41% clearance of soluble tau  and 42–47% clearance of insoluble tau  in the immunized mice. Furthermore, Aβ burden was reduced by 84% overall . These benefits were associated with reductions in microgliosis and microhemorrhages. In summary, prophylactic tau immunization not only prevents tau pathology but also Aβ deposition and related pathologies in a sustained manner, indicating that tau pathology can promote Aβ deposition, and that a short immunization regimen can have a long-lasting beneficial effect. Extensive work by numerous investigators suggests that Aβ pathology may lead to tau pathology2. However, it is interesting to note that reanalysis of a large number of human AD and control brains of various ages with phospho-specific tau antibodies revealed that phospho-tau immunoreactivity is generally detected in control brains prior to Aβ deposition3. This unexpected finding suggests that tau pathology may precede Aβ pathology in AD, at least in certain individuals, although it is of course unclear if these subjects would ever have developed the disease. Importantly, Aβ plaque clearance has had limited effect on tau pathology in the Aβ immunotherapy trials (for review see4), which emphasizes the need for therapy that specifically targets this other major hallmark of the disease. Numerous reports by us and others have shown the feasibility of tau immunotherapy35, and several clinical trials have been initiated (for review see36). However, all these studies were conducted in mice or related culture models that have only tau pathology but no Aβ pathology. Tau antibody immunization in Aβ plaque models has been reported to improve cognition and clear certain Aβ species while increasing Aβ plaque burden37, or when using a different tau antibody in a different Aβ model provided no cognitive benefits and increased mortality38. A few prior studies have reported toxic effects of active tau immunization in mice when very strong adjuvants are being used40.Alzheimer’s disease (AD) is characterized by accumulation of extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles (NFT), and extensive synaptic loss leading to progressive cognitive impairment and eventually dementia. NFT are primarily composed of filaments of aggregated hyperphosphorylated tau protein41. The PS1 and APP mutations individually cause familial AD and the tau mutation leads to frontotemporal dementia, which is an Aβ negative tauopathy. The 3xTg mice develop age-dependent and region-specific Aβ and tau deposits that mimic the disease progression in humans. They have previously received Aβ immunotherapy, which cleared Aβ plaques and rescued early but not late hyperphosphorylated tau aggregates42. More recently, a couple of studies have reported on the effect of tau antibodies on tau and Aβ burden in 3xTg mice44. A single injection of AT8, a phospho-tau antibody was shown to transiently reduce tau pathology without affecting Aβ pathology43. More recently, multiple injections of another tau antibody acutely reduced tau and Aβ pathology in their early stages44. Here, we report that immunization of this 3xTg model with Tau379–408 from 3–6 months of age, with animals killed at 22 months for analysis, resulted in a robust tau antibody response and long-term clearance of not only tau aggregates but also associated Aβ plaques.One of the few models with both Aβ and tau deposits, is the triple transgenic (3xTg) mouse model, which harbors a presenilin 1 mutation (PS1/M146V) knock in allele, as well as the Swedish mutation of the amyloid precursor protein (APPSwe), and tau P301L mutation transgenes5.Phosphorylated tau peptide, Tau379–408, was synthesized and purified at the Keck facility  as described previously41. A breeding pair of homozygous mice and another pair of wild-type (wt) mice of the same mixed strain background (C57BL6/129 SVJ) were graciously donated by Frank LaFerla . These mice develop plaque and tangle pathology in AD relevant brain regions . Wt mice from the same background were used as a control. The mice were housed in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved facilities. All mouse care and experimental procedures were compliant with guidelines of animal experimentation and were approved by the Institutional Animal Care and Use committee at New York University School of Medicine.The treatment was performed in 3xTg transgenic mice expressing knock-in mutation PS1/M146V combined with APP/K670N, M671L and MAPT/P301L transgenesrd immunization; T2-T5: 2, 5, 8 and 11 months after the 4th immunization, respectively; Tf: 16 months after the 4th immunization). At 22 months, their brains were extracted for analyses. The mice went through several behavioral tests in the two months prior to killing. At the end of study, a few mice had died in each of the four groups except in the immunized 3xTg groups, in which all the mice survived the experimental period . Measured parameters were distance traveled (in centimeters), mean resting time, and velocity [mean (Vmean) and maximum (Vmax)] of the mouse.A circular open field activity chamber (70 cm in diameter) was used to measure exploratory locomotor activity over 15 min45. Mice were evaluated by examining their ability to traverse a narrow beam to enter a goal box. The animals were placed on a wooden beam  that was suspended 30 cm above a soft foam cushion by two identical columns. At each end of the beam was attached a shaded goal box. Habituation consisted of placing the mouse on the middle of the beam for 60 s. Subsequently, in four successive trials, the number of foot slips before falling or reaching the goal box was recorded for each mouse. Errors were defined as foot slips and their numbers counted. A mouse that fell off the beam, was placed back on it at the location it fell from.This test measures balance, motor coordination and function integration45 Rotarod 7650 accelerating model; Ugo Basile). Habituation consisted of two trial training sessions to allow the animals to perform at a baseline level. Subsequently, the mice went through three test trials, with 15 min break between sessions. The rotarod was set 1.0 rpm and was gradually raised every 30 s until the mouse fell off or inverted (by clinging) from the rotating rod. The rpm at that point was recorded. To prevent injury, a soft foam cushion was beneath the rod.This test was conducted to measure potential differences in forelimb and hindlimb motor coordination and balance without a practice confound  went through one training session per day for ten consecutive days. In each session, all the arms of the maze were baited with saccharine flavored water, and the mouse was allowed to enter and explore all arms until the eight sugar rewards had been consumed. Spatial learning was assessed by recording the number of errors (entries to previously visited arms) and the time needed to complete each session.An eight–arm radial maze with a water well at the end of each arm was used to evaluate spatial learning45. This symmetrical maze46 is based on the Hebb-Williams47 and Rabinovitch-Rosvold48 tests. Briefly, the key difference is that each end compartment serves as both a start box and a goal box, and the mouse navigates in opposite direction on alternate trials. An advantage of this setup is that it eliminates intertrial handling, which reduces animal stress and thereby gives more reliable data. The barriers are positioned symmetrically in the maze, so that the mouse faces the same turns going in either direction within a given setup. Before testing, the mice were adapted to a water restriction schedule, with 2 h daily access to water. Habituation consisted of two adaptation sessions before the first testing period. In the first such session, all the mice had access to saccharine flavored water in the goal box for 10 min. In the second adaptation session, the mouse was put in the start chamber and allowed to explore the field and enter the goal box, which contained saccharin water reward (0.05 mL). When the mice were consistently traveling between the start and goal boxes, they went through three practice sessions on simple navigational problems, in which one or two barriers were positioned in different regions of the maze to prevent direct route to the goal box. Formal testing consisted of three problems of varying difficulty, starting with the easiest one and ending with the most difficult one. The mice tackled one problem per day and the mice went through five trials to solve it with an inter-trial interval of 2 min. Their performance was scored by the same person for the number of errors (i.e. entries and reentries into designated error zones) and time to complete each trial.This maze consists of a rectangular field  that is covered by a clear Plexiglas top. Two boxes (each 15 × 20 × 9 cm), for the mice to enter or exit are situated at diagonal corners of the maze50, in which the immunogen was coated on to microtiter wells . The binding of plasma antibodies was detected by a goat anti-mouse IgG or IgM linked to a horseradish peroxidase (Pierce), with the enzyme catalyzing a color reaction in the substrate .The antibody response was determined in a 1:200 dilution of plasma using an ELISA assay as detailed previously52. The left hemisphere was snap frozen on CO2 pellets and stored at −80 °C until processed for western blots. The right hemisphere was sectioned coronally (40 µm) from the frontal pole to the cerebellum, and sections were saved into 5 serial series for histological staining with about 40 sections per series. For each stain/antibody, at least half a series (20 sections), spaced equally apart (400 μm) were reacted. Staining was performed at room temperature as described previously52. Briefly, sections were placed in 0.3% H2O2 for 15 min to block endogenous peroxidase activity, and then incubated in mouse-on-mouse (MOM) blocking reagent  to block nonspecific binding for 1 h. Following washes in TBS-Tx, the sections were stained with PHF1 (1:2000 dilution of cell culture supernatant) and MC1 (1:100 dilution of cell culture supernatant) tau antibodies . Adjacent sections were also stained with 6E10/4G8 (1:2000 dilution of 1 mg/ml stock of each antibody) for Aβ deposits. To assess glial response, brain sections were stained with 1) rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP) in astrocytes , and 2) Iba1  to detect microglia. Neurons were stained with cresyl violet using standard procedure as described previously53.After the behavioral testing, the mice were deeply anesthetized with ketamine/xylazine . The brain was then extracted without perfusion and processed as detailed previously54. Briefly, the brain sections were incubated in a mixture of equal volumes of 10% potassium ferrocyanide (K4Fe(CN)6 trihydrate) in distilled H2O and 20% hydrochloric acid (HCl) for 30 min. The sections were subsequently washed with H2O, counterstained with nuclear fast red solution for 10 min, washed again with H2O, dehydrated and coverslipped using Depex mounting media .For staining of microhemorrhages, serial coronal sections of experimental and control mouse brains were mounted on gelatin coated slides and stained with Prussian blue working solution as described previously8. The measurement was the percentage of area in the measurement field (200X) that was occupied by the reaction product . Aβ burden was analyzed as per our standard procedure54, using the StereoInvestigator Program . The area of the grid was 800 μm2 × 800 μm2 and Aβ burden was measured in one frame per section (640 × 480 μm2 each) chosen randomly within the subiculum region, and five sections spaced 200 μm apart were analyzed. The Aβ burden is defined as the percentage of area in the measurement field (subiculum) that is occupied by the reaction product.Tau and Aβ deposition was analyzed in the subiculum of the brain because of its prominent and consistent such pathology. Tau pathology was quantified blindly in 5 sections per brain spaced 200 μm apart similar to as described previouslyThe assessment of the Iba1 (microglia) stained sections was based on a semi-quantitative analysis of microgliosis in the subiculum . The rating of the GFAP sections was based on the complexity of astrocytic branching in the subiculum .The haemorrhage profiles (hemosiderin stain) were counted, and the average number of Prussian blue-positive deposits in the subiculum was calculated for each brain section.All procedures were performed by an individual blinded to the experimental condition of the study. The accuracy of the findings was verified by two independent observers.3VO4, 1 μg/ml complete protease inhibitor cocktail (Roche) and subjected to a low speed spin  to remove the membrane fraction. For sarkosyl extraction, 1% sarkosyl solution was added to 300 μL supernatant for a final concentration of 1% and then incubated for 1 h at 37 °C. Sarkosyl extracted supernatant and supernatant without sarkosyl were then centrifuged at 100,000 × g for 1 h at 4 °C in Beckman TL-100 ultracentrifuge, and the high-speed supernatants were collected and used for western blot analysis. Sarkosyl extraction results in dissociation of insoluble proteins including aggregated tau proteins. For the insoluble fraction, the pellet was re-suspended in the same volume of buffer without protease and phosphatase inhibitors, but that contained 1% (v/v) Triton X-100 and 0.25% (w/v) deoxycholate sodium. It was then ultracentrifuged at 50,000 × g for 30 min to retrieve a detergent extracted supernatant that was analyzed as an insoluble fraction18.Brains were weighed and homogenized in modified RIPA buffer  polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane that was subsequently blocked in 5% nonfat milk with 0.1% Tween-20 in TBS, and incubated with different antibodies for at least 3 h at room temperature or at 4 °C overnight . Following washes, the membranes were then incubated for 2 h with 1:2000 horseradish-peroxidase (HRP) conjugated goat anti-mouse antibody (Pierce), developed in ECL (Pierce), imaged with Fuji LAS-4000, and the signal quantified with Multigauge.The experimental design was as detailed above. Briefly, transgenic and wt mice of both sexes received short-term prophylactic active tau vaccination at a young age (3–6 months). Control mice received only adjuvant. The mice were bled periodically to determine their antibody titers and they went through behavioral testing at the end of the study (20–22 months). Subsequently, their brains were removed for histological and biochemical analyses of the effects of the vaccination on AD related pathologies, and to monitor potential adverse effects.All data were analyzed with GraphPad Prism 7. Unless specified below, the analysis was performed with an unpaired t-test, two-tailed. Welch correction was used if the data failed a test of equal variance. When the data failed at least two of three normality tests , nonparametric Mann–Whitney test was used. That test was also used for analyzing the astro- and microgliosis. Behavioral data was analyzed with one or two-way ANOVA, depending on the number of factors. When the data failed at least two of three normality tests , nonparametric Kruskal Wallis test was used. Radial arm maze test was analyzed with two-way ANOVA, repeated measures.10. In a pilot study, we noticed substantial mortality in the immunized mice after the 5th immunization, with surviving mice maintaining high antibody titer for more than a year. Hence, the enrolled mice received only four vaccine injections. That short vaccination paradigm did not appear to elicit side effects. As noted in Table  Mice immunized with the Tau 379–408 immunogen in Adju-Phos adjuvant developed a robust IgG response in both male and female 3xTg as well as wt mice compared to controls that received adjuvant alone. Fig. . IgM res34, and observed that Tg tau immunized mice performed better than the Tg control mice. These prior studies include the use of the same tau immunogen. The immunized 3xTg mice and wt mice did not differ significantly from their non-immunized identical control mice in any of the cognitive tasks, and all the groups appeared to have normal motor functions based on our experience with wt mice in these tests (data not shown).The tau immunotherapy did not lead to any significant cognitive improvement in the immunized 3xTg mice, compared to adjuvant treated controls (data not shown). Our analysis however, did not detect cognitive impairments in the 3xTg controls compared to wt controls. Hence no therapeutic behavioral benefits were observed with the immunotherapy as the mice were not impaired. We have used these same tests extensively in other tangle modelsTo determine the consequences of the active tau immunization on hyperphosphorylated tau and pathological tau conformers, brain sections were stained with PHF1 and MC1 antibody, respectively. Pronounced tau pathology was revealed with both antibodies, primarily in the subiculum/CA1 region, which was therefore the focus of analysis Figs  and 3A–D2 (average ± SEM); wt immunized = 376,469 ± 98,775 AUC/mm2).Likewise, western blot analysis revealed a similar clearance of tau in the immunized 3xTg mice. Significant reductions were observed in soluble and insoluble human tau (CP27) in the immunized overall group , which fits with the previously reported limited effect of tau or Aβ pathology on this parameter in this model55.Like many other AD models, 3xTg mice do not have extensive neuronal loss8. Overall, these results support the view that Aβ and tau pathologies are synergistic. Clearing tau leads indirectly to clearance of Aβ and associated pathologies such as microgliosis and microhemorrhages.To assess the potential involvement of activated astrocytes, microglia and microhemorrhage following immunization, histological analysis was performed focusing on the subiculum of the hippocampus, the region with the highest Aβ plaque burden and associated tauopathy. GFAP immunoreactivity was greater in 3xTg mice compared to wt mice  but the immunotherapy did not affect astrogliosis Figs  and 8. L4). Also, a single intrahippocampal injection of a tau antibody reduces early and late tau pathology in 3xTg mice without affecting Aβ deposits43. In that particular study, clearance of tau pathology was acute and transient, observed at 7 and 14 days post-injection but was no longer evident 21 days after antibody administration. It is, therefore, not surprising that Aβ pathology was not affected within such a short timeframe. More recently, tau passive immunization was shown to inhibit not only tau but also Aβ pathology in 3xTg mice that received 6 weekly tau antibody injections at the early stages (12 months) of tau pathology in this model44. In our study, the mice received their first vaccine injection at 3 months, and the last one at 6 months, at which age the mice have minimal if any tau or Aβ pathology. The tau antibodies elicited by the vaccine then prophylactically prevented the development of intraneuronal tau aggregates, which then indirectly attenuated Aβ deposition. Further support for the synergistic effects of these two pathologies can be obtained from their regional colocalization in the 3xTg model. The Aβ deposits are most prominent in the subiculum region of the hippocampus and are surrounded by dystrophic neurites positive for pathological tau protein. It can be inferred that neurons with tau lesions generate more Aβ than healthy neurons that is then deposited near their synaptic terminals. This scenario would then further promote tau pathology and enhanced Aβ deposition resulting in a vicious cycle. Long-term antibody-mediated removal of tau aggregates would therefore be expected to attenuate the progression of the intertwined tau and Aβ pathologies as confirmed by our findings.Our findings indicate that tau immunotherapy can not only lead to clearance of tau pathology but also of Aβ deposits. Importantly as well, the benefits are sustained, lasting at least 16 months following the last immunization. Together, these findings may have major implications for clinical use of this approach. Aβ immunotherapy has previously been shown to reduce tau pathology to a modest extent in mouse models and humans . We were not able to detect such impairments in two different cognitive tests compared to age-matched littermate wt mice. The performance of the wt controls in this study was comparable to our prior reports using these tasks in wt controls of a different strain background but of a similar age54. This further supports lack of cognitive deficits of the 3xTg mice in these tasks. However, studies reporting memory issues in this model used different tests. Also, the 3xTg mice we used may have had less severe pathology than in reported studies. Finally, the mice in our study were older than in the prior report. It is conceivable that age-related memory deficits in wt mice may catch up with Tg deficits at the 21–22 months of age when our mice were tested. We are not aware of other reports assessing cognition in this model in mice older than 15–18 months of age56. Most of these studies report deficits at various ages ranging from 3–5 months to 15–18 months with a few showing no impairment compared to controls at 1–2 months, 3–5 months and 9–11 months. None of the tests in the prior studies were similar to ours, which further complicates comparison.Cognitive impairment has been previously documented in 3xTg mice  only a few mice died during the long experimental period (Table Regardless of the lack of a pronounced behavioral phenotype in the 3xTg mice compared to wt mice of the same strain background, the histological effects of the tau immunotherapy were pronounced and highly significant. These findings suggest that prevention of the development of tau pathology can robustly diminish associated Aβ deposition in mice that are prone to develop such deposits because of APP and PS1 mutations. Remarkably, the therapeutic benefits of the prophylactic immunization were long-lasting, up to 16 months after the fourth and last immunization. Prior active tau immunization studies have not assessed such sustained benefits following the vaccination paradigm. Typically, the final immunization in those studies has been within a month prior to brain analysis. What led us down the path of determining possible long-term benefits of active tau immunization was the unexpected high mortality in this model following the fifth immunization in a pilot study that preceded this comprehensive study. Such catastrophic adverse effects most likely relate to the strong immune response in this particular hybrid strain because 3xTg and wt mice on the same strain background were affected to a similar extent. In our prior studies using this tau immunogen and the same alum adjuvant, we did not observe adverse reactions let alone death in two different tauopathy mouse models that received five or more immunizations57.The immunization did not affect tau levels in wt mice. This is as expected because of the phospho-tau immunogen whose epitope is primarily found in pathological tau. Also, the normal tau in wt mice is primarily cytosolic, whereas tau antibodies within neurons typically bind to pathological tau in endosomal-lysosomal vesicles It remains to be seen if such prolonged prophylactic benefits, not only for blocking tau pathology but also for diminishing Aβ burden, will hold up in humans receiving active tau immunizations. It is unlikely that this will be clarified in ongoing active tau immunization trials as the enrolled subjects are likely to have already substantial Aβ deposits, whose development may have plateaued because of synaptic loss. As in our study, prophylactic therapy will likely be required, which could be assessed first in individuals with genetic mutations that are known to cause AD or related tauopathies. However, because the extent of tau pathology correlates much better with cognitive deficits than Aβ burden, tau immunotherapies are likely to provide clinical benefits at later stages of AD than treatments that directly target Aβ.Supplementary Figures - Original blots