Mitochondria have a central role in cellular metabolism; they are responsible for the biosynthesis of amino acids, lipids, iron–sulphur clusters and regulate apoptosis. About 99% of mitochondrial proteins are encoded by nuclear genes, so the biogenesis of mitochondria heavily depends on protein import pathways into the organelle. An intricate system of well-studied import machinery facilitates the import of mitochondrial proteins. In addition, folding of the newly synthesized proteins takes place in a busy environment. A system of folding helper proteins, molecular chaperones and co-chaperones, are present to maintain proper conformation and thus avoid protein aggregation and premature damage. The components of the import machinery are well characterised, but the targeting signals and how they are recognised and decoded remains in some cases unclear. Here we provide some detail on the types of targeting signals involved in the protein import process. Furthermore, we discuss the very elaborate chaperone systems of the intermembrane space that are needed to overcome the particular challenges for the folding process in this compartment. The mechanisms that sustain productive folding in the face of aggregation and damage in mitochondria are critical components of the stress response and play an important role in cell homeostasis. Mitochondria have a central role in cellular metabolism; they are responsible for the biosynthesis of amino acids, lipids, iron–sulphur clusters and regulate apoptosis. Mitochondria have a central role in cellular metabolism; they are responsible for the biosynthesis of amino acids, lipids, iron–sulphur clusters, haem, and lipids. In addition, they are crucial in cellular signalling pathways and apoptosis.S. cerevisiae and 13 proteins in humans.4 These proteins are all core subunits of the electron transfer complexes located at the inner mitochondrial membrane. Approximately 99% of mitochondrial proteins are encoded by nuclear genes and are synthesized as protein precursors in the cytosol. Subsequently they are targeted to the surface of mitochondria and then sorted within their specific sub-mitochondrial compartment.5 The mitochondrial proteome contains about 1000 proteins in yeast and 1500 proteins in humans, fulfilling the mitochondrion's multiple functions.1,2 Proteomic studies have led to the functional classification of these proteins in yeast and more recently in human mitochondria. An inventory of a little over 1000 genes encoding the mammalian mitochondrial proteome, known as MitoCarta, was created in 2008.4 This was updated in 2015 to include a total of 1158 human or mouse genes leading to MitoCarta2.0.6 The latest MitoCarta release in 2020 includes 1136 genes with information on the submitochondrial localisation and the mitochondrial functional pathways they belong to.7Mitochondrial biogenesis requires the integration of two genomes, the nuclear and the mtDNA genome. Despite the plethora of mitochondrial functions, the mitochondrial genome only codes for a small set of proteins, namely 8 proteins in the yeast 7 Despite making up a small proportion of the mitochondrial proteome, proteins of the outer membrane and the IMS play a crucial role in the numerous functions of the mitochondrion. These include, apoptosis, phospholipid biosynthesis, haem biosynthesis and critical communications with the cytosol and other organelles. In addition, although the IMS is the smallest mitochondrial subcompartment, it encompasses a very wide range of protein import mechanisms. Protein import is pivotal for the mitochondrial intermembrane space (IMS) since all IMS proteins are encoded by nuclear genes.2,8According to the inventory, the majority of the proteins are assigned to the matrix (46%) and the inner membrane (32%). 10% are annotated as outer membrane proteins whilst only 5% are designated as intermembrane space (IMS) proteins.In this review we discuss the import pathways required to accommodate for the different types of proteins that are imported into mitochondria. Furthermore, we highlight the types and structural features of mitochondrial protein targeting signals that underpin the different import pathways. We then focus on the IMS, which is the only mitochondrial compartment housing a redox modification machinery integral to the import process. We discuss how this machinery works and how it facilitates a chaperone-assisted folding process in this compartment. Finally, we discuss the various chaperone systems in the IMS which are critical for sustaining an efficient folding environment in this compartment.2.9 The precursors undertaking a specific import pathway are guided by peptide targeting signals that are variable in length and their chemical properties and, in some cases, not very well understood. In the next paragraphs, we will summarise the main protein import pathways in terms of the components involved; the energetic requirements and the mechanism of interactions that ensure the efficiency of the protein import process which is key to mitochondria biogenesis.Several import pathways are needed to accommodate the different types of proteins that are imported into mitochondria . Each of2.110 This sequence is commonly referred to as the matrix-targeting signal (MTS), and will ultimately lead to the translocation of the protein into the mitochondrial matrix.11Cytosolically made proteins encoded by nuclear genes but targeted to mitochondria for their function usually require a system of mitochondrial-linked chaperones and a targeting sequences that determine the protein's endpoint within the mitochondria. Proteins destined for mitochondria typically are translocated as preproteins; preproteins typically contain an N-terminal amino acid presequence that usually forms an α-helix with positively charged residues on one side.12 Upon close proximity with the outer membrane of the mitochondria, the preprotein is typically recognised by the membrane-bound receptors Tom20 and Tom22, both of which form part of the main translocase of the outer membrane (TOM) complex, together with Tom40, the main pore-forming subunit of the TOM complex.2 In addition to Tom20, Tom70 is one of the first identified import receptors of the mitochondrial OM.13 Both Tom20 and Tom70 have their own specific preference for substrate, alongside the fact they are non-essential but have the ability to rescue each other's function.14 Tom40 acts as the main protein import channel and is encoded by an essential gene.15 Tom40 is endowed with the capacity to allow passage of almost all proteins that are translocated to mitochondria, and it contains a specific region for the binding of different types of preproteins. Also included within the TOM complex are the subunits Tom5, Tom6 and Tom7, all of which are encoded by non-essential genes  for the optimisation of the function of the complex.16–18 The matrix targeting signal binds to a groove of Tom20 that recognises its hydrophobic residues;19 recognition by Tom20 leads to the binding and translocation through the outer membrane via Tom40;20 the preprotein then interacts with the presequence translocase of the inner membrane known as TIM23. This interaction between the preprotein and TIM23 drives preprotein translocation across the inner membrane, in partnership with membrane potential (Δψ).21 Coupled to the TIM23 complex is the presequence translocase-associated motor (PAM), containing the mitochondrial version of heat shock protein 70 (mtHsp70). This motor promotes the unidirectional movement of preproteins into the mitochondrial matrix in an ATP-dependent manner.22 Following successful import, the positively charged presequence is cleaved by the mitochondrial-processing peptidase (MPP).23,24 Additional processing enzymes are then available to stabilize and fold the protein into its active form. Examples include: the removal of destabilizing amino acids by 55 kDa intermediate cleaving and octapeptidyl peptidases (Icp55 and Oct1 respectively);25 and correct folding via the Hsp60–Hsp10 chaperonin complex.26The preprotein is carried within the cytosol in an unfolded state maintained by the cytosolic chaperones Hsp70 and Hsp90.2.227 Once stalled within the TIM23 complex, they are then released laterally into the inner membrane with the help of a small hydrophobic protein known as Mgr2.28If a protein is destined for a location other than the matrix, it will usually include either additional targeting information on top of the matrix targeting signal or an entirely different targeting signal. Some proteins that are destined for the IMS interact first with the inner membrane and are then released into the IMS. In this case, they contain a bipartite targeting signal made of a matrix-targeting sequence followed by a specific hydrophobic sorting signal. These proteins are initially targeted to the TIM23 complex following the matrix-targeting route. However, when in the TIM23 complex they get arrested by the small hydrophobic segment which functions as a stop-transfer signal (stop transfer pathway).29–31A separate route for insertion of inner membrane proteins exists for very hydrophobic proteins that are subunits of the electron transport complex of the inner membrane and are encoded by the mitochondrial DNA. These proteins are synthesised in the matrix and inserted into the inner membrane form the matrix side by the OXA insertase.32 The insertion of these proteins in the inner membrane is governed by the carrier pathway.33 In this case, the imported proteins do not contain a presequence, but instead internal peptide sequences found within the more hydrophobic areas of the transmembrane segments of the protein play the role of targeting signals. Proteins with internal signals are carried by Hsp70 and Hsp90 to the outer membrane where they are recognised by the receptor Tom70.12 Following their release from the cytosolic chaperones, proteins travel into the intermembrane space via the Tom40 channel, presumably in a looped formation.34 Small intermembrane space chaperones known as small TIM chaperones (Tim9 and Tim10 or Tim8 and Tim13) then bind – in a heterohexameric complex – to the protein to prevent any aggregation occurring.34–37 Here, the proteins are then guided by the chaperones to the translocase of the inner membrane TIM22 complex. The TIM22 complex then inserts the proteins in a process that depends on Δψ, thus producing a multi-spanning protein within the inner membrane.38 The insertion of Tim23 is like the carrier pathway as it also uses small TIM chaperones to allow transport across the intermembrane space, albeit with the non-essential chaperones Tim8 and Tim13.39–41 An example of a preprotein lacking an N-terminal presequence is the ADP/ATP carrier (AAC), as targeting information to the inner membrane is instead found within its three modules.42A major part of inner membrane proteins are helical multispanning proteins that belong to the family of solute transporters of the inner membrane. These have typically six membrane spanning segments and are responsible for the transport of small metabolites across the inner mitochondrial membrane.2.343 β-Barrel proteins are characteristic prokaryotic proteins. Interestingly, these proteins are also found within eukaryotic mitochondria and chloroplasts, making their lineage unique to the rest of the cell.14 There are three main subunits found within the SAM machinery: Sam50 (the main component), Sam35 and Sam37. Mitochondrial preproteins access this machinery by interaction and translocation into the intermembrane space via the TOM complex's Tom70 receptor and Tom40 channel. Here, the proteins interact with the small TIM chaperones, allowing for the integration of the precursor proteins into the SAM complex, where they are subsequently inserted into the outer membrane.44The insertion of outer membrane proteins also involves the TOM complex, amongst other complexes found within the outer membrane. One such complex is known as the sorting and assembly machinery (SAM) complex and is used for the insertion of outer membrane β-barrel proteins.45 Single-spanning proteins with an N-terminal anchor and multi-spanning membrane preproteins are able to bypass the Tom40 channel and be imported into the outer membrane directly via the MIM complex, usually in partnership with the Tom70 receptor.46 Proteins Mim1 and Mim2 are crucial for the normal function of the complex; complete absence of Mim2 has been shown to lead to the impairment of mitochondrial protein import, defects in mitochondrial morphology and problems in the assembly of the TOM complex.47Although β-barrel proteins are exclusively localised in the outer and not the inner mitochondrial membrane, the outer membrane also houses α-helical membrane proteins. These are integrated in the OM by a dedicated machinery, the mitochondrial import complex (MIM complex).2.448 Bipartite sequences are targeting signals – much like matrix targeting signals – albeit with an added transmembrane-domain-like hydrophobic region following the MTS as discussed in the previous section. These preproteins following the stop transfer pathway are subject to a double cleavage (i) by the mitochondrial processing peptidase (MPP) to remove the MTS and (ii) by the specific intermembrane space protease (IMP) that removes the stop transfer hydrophobic domain –to discharge the mature protein into the intermembrane space. This pathway is fuelled solely by mitochondrial membrane potential, and does not require ATP-hydrolysis in contrast to the matrix targeting pathway where ATP hydrolysis in the matrix is needed.11,49–51All proteins destined for the intermembrane space are encoded by nuclear genes, and thus are synthesised and translocated from the cytosol. For these proteins to reach the intermembrane space, they can include one of two unique properties: (1) they can contain a bipartite sequence as previously discussed, or (2) they can be retained in the IMS by an interaction with the mitochondrial intermembrane space import and assembly protein 40 (Mia40).3C or CX9C – which later become oxidised, forming strong disulphide bonds necessary for intermembrane space protein maturation. This discovery was based upon the presence of internal disulphides found within the TIM intermembrane space chaperones.36,52 This specific pathway is known as the MIA  pathway  is dependent on their interactions with Mia40, albeit without the requirement of their cysteine motifs.11 Tim22 is a translocase that does not have any CXnC motifs but still relies on Mia40 for its correct import and insertion within the inner membrane.10 These interactions display the importance of this single oxidoreductase protein on the regulation and maintenance of mitochondrial intermembrane space proteins.A very substantial part of the intermembrane space proteins contain cysteine motifs – either CX pathway , as prep57 Deletion of this ITS has shown to completely abolish the import of Mia40 substrates to the intermembrane space. The ITS functions by forming a helix with its vital hydrophobic and aromatic residues on the same side as this primed cysteine residue. Here, this region of the helix interacts with and induces the interaction of the substrate with the Mia40 oxidoreductase, ultimately leading to further interaction through disulphide bond formation.57,58It was discovered that a sequence which is not an N-terminal sequence – and is common to many Mia40 substrates – exists within these substrates and is vital for the substrate's specific targeting capabilities to the mitochondrial intermembrane space. This intermembrane space targeting signal (ITS), consisting of 9 amino acids, is necessary for targeting of intermembrane-space-targeted preproteins to Mia40; the ITS functions by priming a cysteine residue within the substrate for effective docking with Mia40's active-site-resident CPC motif.59 Erv1 is a Mia40 substrate with a structure unlike any other Mia40 substrate, containing three specific cysteine pairs that are highly conserved between species ;11,56,59,60 Erv1 is unable to oxidise proteins independently, although it can form a higher ternary complex with Mia40 to achieve this effect.61 Specifically, the first pair of cysteines are involved in the specific interaction of Erv1 with Mia40.62,63 The third pair of cysteines' disulphide bond has a more structural role; interestingly, this same disulphide is recognised by the Mia40 oxidoreductase during the import of Erv1 into the intermembrane space,64 with final folding and maturation of Erv1 occurring with FAD binding.59 Erv1 is a crucial component of the MIA oxidative folding pathway and functions by removing electrons from Mia40 and relaying them to either molecular oxygen or cytochrome c,65,66 thereby producing either hydrogen peroxide (H2O2) or water (H2O) respectively; water is produced from cytochrome c's funnelling of electrons to oxygen linking thus this oxidative folding pathway to the ETC. Specifically, Erv1 functions by collecting electrons  in its own CX2C motif. Here, the electrons are transferred onto FAD, where they can then be relayed to either molecular oxygen or cytochrome c. Although oxygen is generally the final electron acceptor, if electrons are relayed to cytochrome c, alternative structures are known to be final electron acceptors – such as the cytochrome c heme lyase Ccp1.67 In essence, through this specific electron relay, Erv1 recycles Mia40 from a substrate-induced reduced state to a functional oxidised state allowing Mia40 to continue its role as a ‘disulphide donor’. We should note that in human mitochondria, the Mia40 homologue (Mia40 or CHCHD4) translocation to the intermembrane space is dependent on the presence of the human homologue of Erv1 (ALR) and an internal targeting signal; this is due to the lack of an N-terminal membrane anchor that is present in the S. cerevisiae homologue of Mia40.68 This further shows the importance of Erv1 to Mia40's general function within the IMS.When Mia40 donates its disulphide to its substrates it stays reduced. To function again it needs to be recycled back to its oxidised state. This re-oxidation is ensured by a second component of the MIA pathway: a flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidase known as Erv1 .3.70 About two thirds of all mitochondrial proteins are synthesized in the cytosol with a cleavable presequence. Presequences are of variable length, usually 18–50 amino acid residues, although very short (<10 aa) and very long (up to 100 aa) ones have been observed.71,72 An important characteristic of mitochondrial presequences is the formation of an amphipathic a-helix that contains a positively charged face and a hydrophobic face.73 The elements of the amphipathic helix are specifically recognised by receptors and other import components during preprotein translocation by the import machinery.74 Although the components of the import machinery have been well characterised as mentioned in the first part of this review, the cytosolic targeting steps and the proteins involved in these are less well-understood. Specifically, it is still not clear for many mitochondrial proteins whether their targeting signal can be recognised by one or many dedicated targeting factors, the specificity of such targeting factors and their interplay with the outer membrane receptor subunits that decode the targeting signals.Mitochondrial targeting signals can be considered as ‘post codes’ that are necessary and sufficient when added to proteins for their submitochondrial localisation.75 Tom20 has the monumental task of recognising roughly up to 1000 mitochondrial proteins, from non-mitochondrial ones, and recognising at least four different classes of precursor proteins with distinct targeting signals following various import pathways in the organelle.76,77 The import machinery not only functions as recognitions stations for the mitochondrial targeting signals but also as channels and driving forces for the translocation of the preproteins (74The main entry gate of precursor proteins to mitochondria is the translocase of the outer membrane (TOM) complex. The TOM complex consists of multiple subunits; Tom20 has been identified as the general receptor for protein sequences while Tom70 may also have a role in signal recognition.proteins .743.171 A comparative analysis of the N-proteome of mitochondria from mouse, human and yeast revealed that although the N-terminus sequence is poorly conserved, properties such as the length, charge and cleavage site are well conserved.78 These presequences are usually removed in the matrix, after import, by the matrix processing peptidase (MPP). MPP cleavage typically occurs at two amino acids C-terminal to an arginine (R-2).24,71The majority of the matrix proteins contain N-terminal cleavable mitochondrial sequences MTS, . The MTS19,79 Previous analyses into the MTS consensus motif  to drive the precursor protein across the inner membrane and into the matrix.80,89,94,96After recognition the presequence transverses the TOM complex by consecutively binding a number of binding presequence-binding sites spanning the channel formed by the β-barrel protein.71,97 It is a heterodimer consisting of two subunits: Mas1 which is the active site and Mas2 which contains a glycine-rich loop which plays an important role in presequence recognition.98,99 Although there is not a conserved cleavage site motif, most cleavage sites have an arginine residue situated two amino acids before the C-terminus  of the presequence.71 Another element that helps MPP recognise presequences for cleavage is an aromatic residue, such as phenylalanine or tyrosine, at position +1.97,100 MPP is not the only peptidase that can cleave N-terminal presequences as recently the human insulin-degrading enzyme (IDE) and its yeast homologue Ste23 have been linked to MTS cleavage and clearance.101 Although IDE is mainly found in the cytosol, an isoform resulting from the alternative translation of IDE localises to mitochondria and is believed to have an MPP-like function.102 Ste23 interacts with Cym1, an established peptide-degrading enzyme in the matrix to degrade presequence peptides and promote protein maturation.101 Presequence cleavage is not only important for protein maturation but also for cell viability. If MPP is impaired the preproteins will not mature and thus the accumulation of unfolded proteins might lead to aggregation in mitochondria.101,103However, the protein is not functional yet and reaches its mature form after the presequence gets cleaved by the MPP. MPP is a soluble matrix protein that processes the bulk of N-terminal presequences.3.212,42 The carrier protein precursors are guided primarily to the Tom70 receptor of the TOM complex by cytosolic chaperones. Nonetheless, there is growing evidence that Tom70 is not a selective receptor protein strictly involved in mitochondrial protein import and it functions as a versatile protein adaptor that facilitates the interaction of mitochondria with different proteins but also other organelles such as the ER.104 The carrier precursors are inserted into the Tom40 channel in a loop formation with both termini remaining exposed on the cytosolic side.42,77 Subsequently, chaperones are recruited in the IMS-side of Tom40 that facilitate the translocation of the hydrophobic precursors and protect them from aggregating or misfolding in the aqueous phase. These chaperones are the small Tims, Tim9–10 and Tim8–13, that we will discuss in detail in the next section. The transient binding of the importing carrier proteins to the small Tim chaperones is guided mainly by the transmembrane segments of the carrier proteins that function as the internal targeting signals.36,40 The presence of the entire sequence of the carrier proteins is presumably necessary to ensure the correct targeting of these proteins, since truncated versions of carrier proteins may be mistargeted to the matrix due to the presence of cryptic matrix targeting signals that would be rendered non-functional in the context of the entire protein and therefore allow proper targeting to the inner membrane.105 The small Tim chaperone complexes associate with the TIM22 complex of the inner membrane to aid the transfer of the precursor protein for final insertion into the membrane. The TIM22 complex consists of Tim18, Tim22 and Tim54, with Tim18 and Tim54 stabilising the complex. TIM22 also requires the membrane potential in order to facilitate the insertion of the transmembrane segments of the integral inner membrane proteins.38,106–108Most inner membrane proteins belong to a family of solute transport proteins , such as the ADP/ATP carrier, and contain six α-helical transmembrane domains. Unlike the aforementioned cleavable presequences, carrier precursors contain internal targeting elements that remain part of the mature protein.3.310,71 Additionally, there are numerous protein import pathways into the IMS perhaps indicative of its pleiotropic nature. One well-characterised type of targeting signal for the IMS is the bipartite presequence for a small number of IMS proteins. Additionally, for the majority of IMS proteins contain characteristic cysteine motifs48,109 and have their own unique targeting signal. The proteins with bipartite presequences contain an N-terminal mitochondrial targeting signal (MTS) as well as a hydrophobic sorting region. The N-terminal part of the presequence is similar to the cleavable mitochondrial targeting presequences, hence it is positively charged and forms an amphipathic helical structure. The positive domain of the bipartite sequence is recognised by the TOM complex receptors Tom20 and Tom22. This signal then allows the presequence to transverse the Tom40 channel. Once in the IMS side of the TOM complex (trans site), it is recognised by Tom50 that in turn feeds the presequence to TIM23.74 The latter region, known as the stop-transfer sequence, arrests translocation to the matrix inserted as the hydrophobic segment gets “stuck” in the Tim23 channel. The transmembrane domain is then laterally diffused and the presequence is anchored to the inner membrane with the N-terminal peptide facing the matrix. The MPP then cleaves the presequence to facilitate protein maturation.101 There is a secondary cleavage site between the hydrophobic segment and the mature protein facing the IMS. This motif is recognised and cleaved by inner membrane peptidase (IMP).27,110 The IMP is a heterodimer consisting of two different catalytic subunits; the Imp1 and Imp2 that are anchored to the inner membrane by their N-terminal domain. A third protein Som1 was shown to be involved in the IMP complex and was found to be essential for the proteolytic activity of the Imp1 subunit. The IMP complex cleaves the transmembrane segment leading to the release of the mature protein in the IMS.49Although the IMS is the smallest mitochondrial subcompartment, it is vital for mitochondrial function. It constitutes a crucial buffer between the cytosol and matrix through the exchange of lipids, proteins, metals and other cofactors necessary for mitochondrial function, redox regulation and apoptosis.3C and CX9C) that form intramolecular disulphide bonds as a consequence of their interaction with the MIA machinery. These targeting signals are termed mitochondrial IMS sorting signal (MISS) or the IMS targeting signal (ITS). The ITS peptide is usually nine amino acids-long and contains conserved residues that are important for both substrate recognition and folding of the substrate protein. The cysteine residues are usually separated by 3 or 9 amino acids, although some MIA substrates contain cysteines not organised in a specific motif. A comprehensive protein engineering and mutagenesis analysis has defined the consensus motif in the ITS signal, CXX[Hy][Hy]XX[Ar]X, where the hydrophobic residues in position −3 and −4 and an aromatic residue in position −7 are strictly conserved  downstream of the stop-transfer signal. When their presequence is translocated across the outer membrane into the IMS, the HBD domain can fold independently of the presequence. As a result, the mature form of the HBD prevents the backward movement of the presequence and thus the presequence can only move forward into the IMS.S. cerevisiae provided evidence that under H2O2 stress conditions, ribosomal binding sites shifted upstream of the endogenous AUG codon leading to alternative translation.117 Gpx3 localises to the cytosol when translated through its canonical AUG start codon. However, under acute oxidative stress, an isoform of the protein is synthesized, with and 18-amino acid N-terminal peptide, from the translation of a non-AUG codon and is translocated to mitochondria. The N18 peptide is mostly hydrophobic and has positive charges but does not follow any of the conventional protein import pathways into the intermembrane space.118 Intriguingly, the Gpx3 without the N-terminal extension also gets targeted to mitochondria, albeit in lower yields than the N-extended form, suggesting that an as yet uncharacterised targeting signal must be present even in the mature part of the protein.8 Gpx3 is not the only protein that is dually localised in the IMS in addition to its cytosolic localisation. Other such proteins are the thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2) and thioredoxin reductase Trr1, which are both targeted to the IMS by yet unknown targeting signals.119Gpx3 is the major oxidative stress sensor in the cytosol. A ribosomal profiling study in 3.52 β-Barrel proteins make up two of the critical translocases of the protein import pathway (the Tom40 and Sam50 channel forming subunits of the TOM and SAM complex) and their function is therefore essential. They are synthesised in the cytosol like 99% of mitochondrial proteins but do not contain a cleavable targeting signal. Instead, the targeting signal of β-barrel proteins is internal to the protein and remains part of the mature protein. At first, the β-strands on the proteins were believed to be the targeting signal for these proteins but no specific motif was identified.120 However, it was later discovered that the actual targeting signal consists the most C-terminal β-strand called the β-signal;121 the properties of the β-signal have been further dissected to include high hydrophobicity and form a beta hairpin element that can extend to include two adjacent β strands that specifically bind the translocation machinery.122 The β-hairpin is recognised by Tom20 and subsequently translocated through the Tom40 channel. Upon translocation, TIM chaperones in the IMS bind to precursor in order to prevent aggregation in the aqueous sub-compartment. It is then transferred to the SAM complex, which is the central machinery for the β-protein biogenesis pathway. The SAM complex contains Sam50, a β-barrel protein itself, which is integrated in the outer membrane, as well as Sam35 and Sam37 that are peripheral membrane proteins exposed to the cytosol. Sam35 and Sam50 cooperate in the recognition of the β-hairpin motif and direct membrane insertion. The β-signal is then proposed to induce the opening of the lateral gate of Sam50 which leads to the lateral release of the protein to the outer membrane.2,121 Sam37 is understood to support the release of the β-barrel protein from the SAM complex to the OM but the exact mechanism of the release is not understood in great detail.123The outer membrane possesses two types of integral membrane proteins: β-barrel and α-helical proteins. β-Barrel proteins are integrated to the outer membrane by multiple β-strands traversing the membrane while α-helical proteins are anchored to the membrane by one or more transmembrane α-helices.124 They are split into three main protein classes: signal-anchored, tail-anchored and polytopic outer-membrane proteins. Signal-anchored and tail-anchored proteins contain an α-helical transmembrane section at the N- and C-terminus, respectively. The transmembrane domain and its flanking regions function as both a membrane anchor and a targeting signal. The exact targeting signal for polytopic proteins is not known but it may be associated with the multiple transmembrane domains of the proteins. Tom70 is believed to be the receptor for such proteins but it is unclear if they pass through the Tom40 channel for their import.125 The MIM complex is proposed to have a major role in this pathway; it can mediate the transfer of substrates from the TOM complex as well as promote the import and assembly of all types of α-helical proteins , they need to be folded at the 24,127In the mitochondrial matrix, various molecular chaperones have been identified that help newly imported proteins to fold. These belong to the well characterised families of Hsp70 Hsp60 and their cochaperones Hsp40 an Hsp10. One key chaperone in the matrix is for example the mitochondrial heat shock protein (mtHsp70) is an ATP-dependent import motor of the TIM23 complex that not only helps drive protein translocation, but also mediates the folding of the imported substrates.128 Second, the IMS is the most constricted mitochondrial sub-compartment, with local concentrations of proteins very high and an increasing potential for aggregation, particularly for proteins with exposed hydrophobic patches which are common for the unfolded imported proteins. Finally, the extended physical interactions between the outer and inner membranes in contact sites and the internal segregation of the cristae lumen separately from the boundary intermembrane space put further constraints on the folding process in this compartment.By contrast, the IMS presents several unique challenges as a folding environment for the newly import proteins. First, the IMS does not retain its own pool of ATP as the matrix, and in fact there are no ATP-dependent chaperones in the IMS.In this context, we will discuss in this chapter the three main chaperone systems in the IMS. Namely, we will focus on the structure and chaperone function of Mia40 as a holdase, the function of the small Tims as dedicated membrane protein chaperones and finally the chaperone role of the protease Yme1.4.153,56 The most notable difference between the two homologs is that the yeast variant contains a large N-terminal extension of 242 amino acids that tethers the protein into the inner membrane facing the IMS, while this segment is absent in CHCHD4 resulting in the protein being entirely soluble in the IMS. Despite their variance, they share a high sequence identity in the central and catalytically active region of the protein (residues 47–107 in CHCHD4 and residues 290–350 in yeast) which include the conserved cysteine residues in a –CPC–CX9C–CX9C– motif. The Nuclear Magnetic Resonance (NMR) structure of this segment for the human protein (53 Mia40 substrates share a coil–helix coiled coil–helix (CHCH) motif that is imported into mitochondria in a reduced and unfolded form.34,52 The oxidative folding ability of Mia40 depends on the formation of transient intramolecular disulphide bridges between the Cys55 of the CPC motif and specific cysteines of the substrate proteins. Successful binding achieves a rapid disulphide exchange that results in the CHCH domain of substrates being oxidised while Mia40 is reduced. Such exchanges include the Cys45 in the CX9C motif of Cox17,53 and the first cysteine of the CX3C motif in Tim9 and Tim10 that regulates the assembly of the TIM9·10 complex.37,129 Aside from the oxidative function of Mia40, the orientation of the substrate with the correct Cys to the CPC motif was found to be crucial to achieving efficient disulphide exchange and strictly dependent on hydrophobic packing between the ITS and the Mia40 substrate cleft.57 The Mia40 structure revealed several conserved hydrophobic residues on the α-helix opposite the CPC motif made up of residues Leu42, Ile43, Ile49, Trp51, Leu56, Met59, Ala60, Phe68, Phe72, Phe75, Phe91, Met94 and Met98. These residues were collectively shown to be essential for cell survival.53 The hydrophobic collapse of the ITS hydrophobic residues, that make up the solvent-exposed hydrophobic binding cleft of Mia40, stabilise the unfolded substrate in the IMS milieu, with Mia40 functioning as a holdase.57,130 This initial step of substrate binding to Mia40 (and hence its chaperone activity) is independent of subsequent oxidation (i.e. disulphide bonding) steps.57,112 In fact, the hydrophobic interactions dictated by the substrate binding cleft of Mia40 are also involved in the recycling of Mia40 by Erv1. In this reaction, Erv1 binds to Mia40 in a manner that mimics the substrate (‘substrate mimicry model’) and making use of an extended hydrophobic patch in the intrinsically disordered N-terminal of Erv1.62,63 While Mia40 catalyses disulphide bridges that thermodynamically favour α-helical folding for its substrates,131in vitro experiments of Koch et al., with Mia40 and Cox17 proposed an additional proofreading ability for the chaperone by re-shuffling incorrect disulphides within its substrate protein.132As previously described, several IMS proteins are required to go through oxidative folding during maturation by interaction with an oxidoreductase, namely Mia40 in yeast or its human homolog CHCHD4. protein  revealed4.235,36,108 Additionally, the small Tims also assist the integration of β-barrel protein in the OM of mitochondria, making them the only known chaperone system capable of interacting with both types of membrane proteins  the family members include Tim8, Tim9, Tim10, Tim12 and Tim13 in S. cerevisiae and are organised in specific oligomeric assemblies. Tim9 pairs with Tim10 and Tim8 with Tim13. They share a conserved CX3CXnCX3C motif (where n ∼ 15) or two CX3C motifs that are essential for their function.52,129 Tim9 and Tim10 are essential proteins35,133 that share 22% sequence identity and 50% sequence similarity. By contrast, Tim8 and Tim13 are not essential for viability. Following their import into the IMS, they assemble into a thermodynamically stable 70 kDa hexameric complex.37,134 Once assembled, Tim12, an essential subunit of the TIM22 complex binds the Tim9·10 complex to enable (i) substrate insertion into the inner membrane and (ii) dual localisation for the chaperone, in the bulk of the IMS and tethered to the inner membrane in a Tim9·10·12 complex intermediate.35,135 Folding of the unassembled Tim9 and Tim10 requires their oxidation by Mia40 and it is only the oxidised and fully folded Tim9 and Tim10 that can assemble to form a functional heterohexameric Tim9·10 chaperone complex.52,129,134 The secondary structure of the complex is exclusively α-helical, where the two subunits alternate in a 6-fold molecular symmetry to form a closed circular core. Each subunit folds into a helix-loop-helix as disulphide bridges in-between CX3C motifs create a central loop. The whole complex comprises 6 inner and 6 outer helices  with a conserved aromatic-hydrophobic motif that is essential for binding substrate proteins. ATPases are ATP-driven machineries that are characterised by a conserved P-looped AAA domain containing Walker A and Walker B motifs. The AAA domain of Yme1 contains a conserved Walker A motif, with the pattern G-X(4)-GK-[TS].144 Numerous studies show the involvement of the motif in recognising and binding substrates for translocation or degradation. It is also hypothesised that this motif is involved when Yme1 binds substrates to prevent aggregation or mediate protein folding.141Despite the crucial function of Yme1, little was known about is structure until Puchades e domain . As seenin vitro experiments using isolated mitochondria.141,145 Moreover, it was suggested that Yme1 assists with the folding/assembly of Cox2, a subunit part of the catalytic core of cytochrome c oxidase. Thus, supporting that Yme1 acts as a chaperone also in vivo conditions.146 Later research confirmed the role of Yme1 as a folding helper for DHFR in the IMS. Importantly, the chaperone activity of Yme1 was independent from its protein degradation function. The study also identified numerous endogenous substrates that aggregate in the absence of Yme1. These proteins are necessary for the aforementioned functions of the IMS, thus further supporting the importance of Yme1.142 Interestingly, Erv1 was identified as one of the endogenous substrates of Yme1. As mentioned above, Erv1 is an essential component of the oxidative folding pathway. Therefore, Yme1 presumably has a crucial role in a variety of mitochondrial pathways.The AAA domain of Yme1 was first shown to act as a chaperone by binding to unfolded protein dihydrofolate reductase (DHFR) in 142 The cryo-EM structure of Yme1 has given a better picture of the architecture of the protein and the residues involved in substrate binding.138 Nevertheless, the exact mechanism of the chaperone activity of Yme1 still needs to be determined.Despite the growing evidence in support of Yme1 as a chaperone in the IMS, the mechanism of its function is not yet clear. It is hypothesized that Yme1 binds newly imported proteins during their translocation and assists with their folding. Another hypothesis is that the AAA domain of Yme1 binds unfolded proteins and helps keep them in a folding-competent state to enable their refolding. If folding is unsuccessful, then the proteins get degraded by the proteolytic domain of Yme1.The different chaperone systems in the IMS  seem to display specificity against their substrates with no overlap in their function. However, the small Tims interact both with Mia40 as a substrate and they also seem to be subject to quality control by the Yme1. It is not yet very clear whether other proteins may be processed by more than one of the IMS chaperone systems.5.Mitochondria have developed multiple import pathways to accommodate the import of the different types of proteins needed for mitochondrial function. Most mitochondrial sub-compartments have typically one or two major import pathways, but the intermembrane space seems to divert with a very high variety of pathways, some of which are dictated by targeting signals that are still either completely unknown or poorly understood. This is surprising given that the IMS is the smallest mitochondrial sub-compartment, but it is perhaps indicative of the pleiotropic function of the IMS.The emergence of new IMS proteins carrying atypical signals is of great interest as not much is known about targeting signal specificity and its recognition by the various import components. Furthermore, there is growing evidence that proteins like the Mia40, the small Tims and Yme1 have more functions than previously anticipated. Taken together, these might suggest that mitochondria have found ways to evolve in order to recognise novel targeting signals or have developed such alternative import pathways as a backup when mitochondrial function is impaired. Thus, it is important to dissect how these unconventional targeting pathways work in order to better understand mitochondrial function and how it can be altered to prevent disease progression.The authors declare no conflict of interests.


Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis , but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents. Cells coordinate the activity of biosynthetic and catabolic pathways as well as the uptake and secretion of components in order to maintain appropriate molecular composition. In continuously proliferating cells, these processes are coordinated so that all cellular components are doubled during each cell cycle, whereas differentiating cells may change their composition to match their functions . ChangesCells’ total molecular content, that is, dry mass, can be monitored using methods such as quantitative phase microscopy (QPM). Dry mass measurements are critical for understanding cell size and growth regulation, but they are often limited in resolution and, more importantly, they do not inform us about the composition of the dry mass. One approach that overcomes this is stimulated Raman scattering microscopy, which can measure the total amount of proteins and lipids in a live cell . While R2O) and heavy water -based solutions  and for very short durations (10–15 s), thus allowing us to non-invasively monitor the dry mass and dry mass density of the same cell over many generations without compromising cell growth and viability. By definition, the measured dry mass density is independently of the cell’s water content and therefore sensitive toward changes in the relative molecular composition (dry composition), not the overall concentration of dry mass. For example, a decrease in the amount of cellular lipids increases the dry mass density of a cell, as lipids are lower in dry mass density than other cellular components.Here, we introduce a new approach for monitoring single cell’s dry mass , dry volume , and density of the dry mass (i.e. dry mass/dry volume), which we will refer to as dry mass density. Cell’s dry mass, dry volume, and dry mass density can be quantified by comparing the cell’s buoyant mass in normal water , dry volume , and dry mass density (i.e. dry mass/dry volume). Importantly, our approach assumes that all water within the cell is exchangeable between H2O and D2O. Accordingly, our dry volume measurement is distinct from the excluded cell volume detected by measuring cell volume following strong hyperosmotic shocks, which does not remove all water from the intracellular space.In our approach, we consider the buoyant mass of a cell to be dependent on two distinct physical ‘sections’ of the cell, the dry content and the water content. To measure the cell’s dry content independently of the water content, we measure the cell’s buoyant mass in H2O-based solution to obtain a buoyant mass measurement , as are D2O and D2O-based media (1.10 g/ml), the density of water inside the cell is near identical to the density of fluid outside the cell whether the measurement is carried out in H2O or D2O-based solution  by cooling our measurement system down to +4°C to prevent growth and compositional changes. We measured the same cell repeatedly and defined our measurement error as the coefficient of variation during a 1 hr trace. With this approach our measurement errors in dry mass and dry mass density were ~1.3% and ~0.3%, respectively . We did 2O and D2O, and this exchange has to be complete for our measurements to reflect the dry mass and dry mass density of cells. Direct experimental evidence suggests that cells normally exchange their water content within ~1 s , and S-Hela (human adenocarcinoma) cells toward different concentrations of D2O by monitoring cell proliferation with live cell imaging. Under normal culture conditions, all three cell lines can tolerate continuous exposure to up to 30% D2O without changes to proliferation rate  for L1210, BaF3, and S-Hela cell populations (R2~0.99), but lower and more variable correlations in mitosis and cytokinesis (R2~0.5–0.9)  . TogetheCells’ dry mass and buoyant mass will correlate if dry mass density does not change, assuming that measurements are carried out in normal cell culture media or other water-based solution with density close to water see . Indeed,We then examined dry mass and dry mass density behavior(s) in mitosis. We detected mitotic stages based on acoustic scattering from the cell, which reports approximate mitotic entry and the metaphase-anaphase transition . We alsoThe early mitotic dry mass and dry mass density changes were not observed uniformly across all wt and FUCCI L1210 cells . ApproxiWe also monitored the dry mass and dry mass density of BaF3 and THP-1 (human acute monocytic leukemia) cells through mitosis. The BaF3 cells displayed a large degree of cell-to-cell variability in mitotic behavior. While some individual BaF3 cells lost ~4% of their dry mass during early mitosis, on average the BaF3 cells did not lose dry mass in mitosis but growth rather stopped during early mitosis, which was followed by a rapid recovery at metaphase-anaphase transition . In THP-S-trityl-L-cysteine (STLC) and validated mitotic arrest by quantifying the fraction of mitotic cells using DNA and p-histone H3 (Ser10) labeling -amiloride (EIPA). We first validated that EIPA prevents mitotic cell swelling by monitoring approximate intracellular water volume  and STLC, respectively . NotablyWe then investigated if inhibition of lysosomal exocytosis prevents the dry mass and dry mass density changes taking place in mitosis. For this, we utilized 10 µM brefeldin A, an inhibitor of vesicle-mediated protein trafficking and exocytosis , and 15 We then monitored the dry mass and dry mass density of FUCCI L1210 cells in the presence of brefeldin A or vacuolin-1. Both chemicals decreased the mitotic changes to cells’ dry mass and dry mass density  without Here, we have developed a method using the SMR to monitor the dry mass, dry volume, and dry mass density of suspension grown cells over long periods of time with high precision. To the best of our knowledge, this is the first method capable of non-invasively measuring cell’s dry volume or dry mass density. We anticipate several uses for this method. First, our dry mass density measurements provide an orthogonal approach for studying the macromolecular composition of cells. While dry mass density measurements lack molecular specificity, they are non-invasive, quantitative, and have high temporal resolution. Together, these qualities make our measurements valuable for studying the dynamics of molecular composition changes, especially in situations where rapid changes cannot be easily resolved by more typical population level measurements, such as mass spectrometry. Second, our method provides an additional approach for measuring dry mass. QPM has several advantages over our method, including higher throughput and suitability for adherent cells, but our method is comparable in precision and has high temporal resolution. Our method is also not limited by phototoxicity. This enables us to observe dry mass dynamics that may be difficult to capture with other methods. Third, the ability of our method to measure cellular dry volume could enable precise, specific, and non-invasive quantifications of single-cell water content, provided it is coupled with a measurement of total cell volume.Using our method, we have expanded our previous study of mammalian cell growth dynamics in mitosis. Importantly, the mitotic dry mass dynamics differ from our previously reported buoyant mass dynamics due to a transient change in dry mass density in early mitosis . This fi2O exposure  Lysosomal exocytosis acts in plasma membrane repair , and lys2O-based media for experiments and the D2O-based media was made to an identical composition using RPMI media powder  and heavy water . D2O-based media pH was adjusted to 7.4 with HCl and NaOH, and the media was filtered with 0.2 µm filter. To minimize cell exposure to heavy water in the SMR experiments, the D2O-based media was diluted with the normal H2O-based media to contain 50% heavy water. All experiments were started using cells growing exponential at a confluency of 250.000–500.000 cells/ml.L1210, BaF3, THP-1, and S-Hela cells were cultured in RPMI media  supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, and antibiotic/antimycotic. This served as the HAll cell lines were tested negative for mycoplasma. L1210 cells were obtained from and authenticated by ATCC. BaF3 cells were obtained from and authenticated by RIKEN BioResource Center. THP-1 cells were kindly gifted by the laboratory of Jianzhu Chen from Massachusetts Institute of Technology, and S-Hela cells were kindly gifted by the laboratory of Kevin Elias from Brigham and Women’s Hospital. No additional authentication was done for THP-1 and S-Hela cells.2O was measured by imaging cell confluency on a plate over ~3 days. Confluency was monitored using the IncuCyte live cell analysis imaging system by Sartorius. Imaging was carried out every 3 hr using 4× objective and confluency was analyzed using IncuCyte S3 2017 software’s standard settings. The confluency data were then used to calculate confluency doubling time, which was used as a proxy for cell cycle duration. For validations of cell morphology in mitosis, cells were imaged every 2 min with 20× objective using the IncuCyte. Metaphase-anaphase transition was detected by simultaneous imaging of the FUCCI cell cycle sensor on FITC channel. The example images were overlaid and processed in the IncuCyte S3 2017 software.Cell population growth in medias with increasing amounts of DChemical treatment concentrations are indicated in main text and supplier details can be found in the key resources table. For SMR experiments, the cells were not pretreated with the chemical inhibitors, and all treatments started at the beginning of each SMR experiment. The treatment time until mitosis therefore varies between experiments, with typical range being 2–4 hr. Apart from EIPA treatments, all SMR experiments with chemical inhibitors lasted only for one cell division.For cell cycle synchronization in G2 and mitosis, cells growing at ~400.000 cells/ml confluency were first arrested in G2 using 5 μM RO-3306 treatment. For L1210 and BaF3 cells, this treatment lasted for 6 hr; for THP-1 cells this treatment lasted for 20 hr. The cells were then washed twice with cold PBS and moved into fresh media containing either 5 μM RO-3306 (for G2 arrest) or 5 μM STLC (for prometaphase arrest). For L1210 and BaF3 cells, this treatment lasted for 1 hr; for THP-1 cells this treatment lasted for 2 hr. After this, the cells were either fixed for cell cycle analysis or immunolabeled with anti-LAMP-1 antibody in drug containing media. Cell cycle arrest in G2 and mitosis was validated using flow cytometry-based detection of DNA and p-histone H3 (Ser10) labeling, as detailed before . ResultsAB_657536). The cells were moved to fresh media  and incubated with the antibody at 1:50 dilution for 40 min on ice, after which the cells were washed twice with fresh media and either imaged or measured using flow cytometry. For antibody isotype control, we used rat monoclonal IgG2a kappa Isotype Control antibody conjugated to Alexa Fluor 488 . For THP-1 cells, the immunolabeling protocol was otherwise identical, but the immunolabeling was carried out with mouse monoclonal anti-LAMP-1 antibody conjugated to Alexa Fluor 488  or with corresponding isotype control (RRID:AB_470230).In L1210 and BaF3 cells, the cell surface localized LAMP-1 protein was detected using immunolabeling with rat monoclonal anti-LAMP-1 antibody conjugated to Alexa Fluor 488 . For microscopy, we used DeltaVision wide-field deconvolution microscope with standard filters , 100× objective and an immersion oil with a refractive index of 1.516. For each image, we collected 15 z-slices with 0.2 μm spacing, deconvolved the images using SoftWoRx 7.0.0 software and selected a middle z-slice for visualization. For flow cytometry-based detection of LAMP-1, DNA was not stained and LAMP-1 immunolabeling was detected using BD Biosciences flow cytometer LSR II HTS with excitation laser at 488 nm and emission filter at 530/30.Image quantifications were carried out manually from single z-layers by cropping out each cell and calculating the average LAMP-1 labeling intensity per cell in ImageJ (version 2.0.0-rc-69/1.52 p). z-layers with intracellular labeling were rare and not used for analysis. Cells were classified as interphase, early, or late mitotic cells based on their DNA morphology. After this, the background signal was removed from the cell signals, and the LAMP-1 labeling of each cell was normalized to the average intensity observed in all interphase cells within the same image .2O-based media and the other side with D2O-based media. For end-point single-cell measurements, the cells are first loaded on the H2O-based media side of the device at ~300.000 cells/ml concentration. A cell is then flown through the SMR to obtain a measurement in the H2O-based media. On the other side of the cantilever the cell is diluted in D2O-based media, which is in excess to the H2O-based media to maximize dilution. In a typical end-point measurement, the cell spends 5–8 s in the D2O-based media. The cell is then flown back through the SMR to obtain a second measurement, this time in the D2O-based media. After this, both sides of the cantilever are flushed with fresh media and cell(s) and the cycle is repeated. For single-cell monitoring, the protocol is the same as for end-point measurements, except that the cell is maintained for ~55 s in the H2O-based media and ~15 s in D2O-based media. Time in D2O-based media is kept longer than in end-point measurements, as this increases the long-term stability of single-cell trapping. Importantly, the SMR baseline frequency is recorded prior and after every cell measurement. The SMR baseline frequency is directly proportional to media density, which allows us to adjust each measurement for local mixing of H2O and D2O-based medias around the cell.Basic SMR build and operation was identical to that reported before . The con2O or D2O) inside the cell (The two buoyant mass measurement (SMR frequency response to a cell flowing through the cantilever) and media density measurements (SMR frequency baseline immediately before and after the cell flows through the cantilever) were used to calculate dry mass, dry volume, and dry mass density according to the cell . In ordethe cell .The precision of dry mass and dry mass density measurements was evaluated by repeatedly measuring the same, non-growing particle. As there are many sources of noise, three types of non-growing particles were used: a fixed L1210 cell measured at +37°C (fixation in 4% PFA in PBS for 10 min followed by a wash with PBS), a live L1210 cell measured at +4°C, and a 10 µm diameter polystyrene bead measured at +37°C. Each particle was tracked for dry mass and dry mass density similarly to that described above for live cells. For each experiment, 1 hr long section of the trace was used to calculate the measurement precision, presented as the coefficient of variability. When evaluating experiment-specific measurement noise , we fittApproximate intracellular water volume of FUCCI L1210 cells was measured in the SMR by growing the cells in culture media containing 40% OptiPrep Density Gradient Medium (Sigma-Aldrich). The OptiPrep containing media has a solution density which is closer to cell’s dry mass density than the density of water, thus making the SMR-based buoyant mass measurement predominantly sensitive toward intracellular water volume . Apart fFor analyses of mitotic dry mass and dry mass density dynamics, each cell trace analysis was limited to data starting from 1 hr before mitotic entry and ending at 2 hr after mitotic entry or at cell division. The very last datapoint prior to abscission was excluded in order to avoid analysis of cells during the final abscission. The traces were then normalized to their average value observed in the 1 hr period before mitotic entry, and the traces were overlayed. When plotting the dry mass, dry volume, or dry mass density mean ± SD values for each control and/or treatment condition, the moving and locally weighted average was plotted together with identical length moving standard deviation. For quantifications of dry mass, dry volume, or dry mass density changes in early mitosis, we identified the time point in mitosis where, on average, each control/treatment reached the minimum dry mass. The average of five datapoints at the maximal effect size was then compared the average of five datapoints immediately prior to mitotic entry (inclusive of the indexed mitotic entry timepoint). The difference between these two values is defined as ‘early mitotic change’. Note that the maximal effect size was always achieved prior to metaphase-anaphase transition. Same smoothing and quantification approach was also used for processing the intracellular water volume measurements. For single-cell examples, the cut-out traces were smoothened using LOESS smoothing.The mode of cell growth was analyzed by comparing an exponential and a linear fit to the raw dry mass data in interphases. AIC was used to define which fit is better . Dry masThe statistical tests used, the results of these tests and detailed information about replicate numbers are indicated in each figure and figure legend. All statistical tests were carried out using Origin (version 2021b) software. Appropriate sample sizes were not computed before experiments and sample sizes were defined by practical considerations . We did not blind-control experiments, as most work was carried out by a single individual. When trapping a cell in the SMR for dry mass and dry mass density measurements, we trap randomly the first normal-sized cell that flows through the SMR after cells are flushed into the system.All dry mass and dry mass density traces of live cells, including those where cells are treated with chemical inhibitors, are available in  The authors measure dry mass, dry volume and the density of the dry mass in growing and proliferating mammalian cells at high temporal resolution and with high precision. Using this method to study mitotic cells, the authors show that some cells lose dry mass early in mitosis by a mechanism involving exocytosis. This work improves upon the authors' method to measure the mass of single cells and its thought-provoking conclusion is that dividing cells 'clean out' their contents to give the daughter cells a fresh start. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Matthieu Piel (Reviewer #1).Thank you for submitting your article "Single-cell monitoring of dry mass and dry density reveals exocytosis of cellular dry contents in mitosis" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) All the reviewers request that you clarify the definition of dry mass, dry density, dry mass density, and dry volume. Below you will see the comments of each reviewer on this point, which should help you to clarify these terms.2) A mathematical model could help to clarify the interpretation of your results. Referee 3 suggests a simple mathematical model that would roughly estimate how the cell composition  should change and what the composition of the lost (secreted) components should be to provide the observed changes to the dry mass and density, given the existing information on average cell composition and the densities of different biomolecules .3) Some of the drug treatments require more replicates to provide more conclusive answers. In particular, Figure 4E-F: Given the SD for barasertib and EIPA cells, the authors need more than ~6-10 cells to draw conclusions based on treatment effects. That barasertib treatment does not affect dry mass and dry density changes is not yet convincing.Other comments:1) The authors show that their measure does not depend on the time spent in D2O, which is important. But it means that the water needs only a few seconds to exchange. Is that expected given the permeation of water through the plasma membrane and the size of the cell? I did not make the calculation, but it seems a bit short. Can the authors give also the theoretical estimate based on the permeation of water through the membrane?2) For the measure of the exponential growth rate, the authors could use the same approach as in Cadart et al. Nat Com 2018, which might be more robust than fitting on the single cell growth curve: measuring the growth speed (dm/dt) on short periods of time all along the cell growth trajectories and for all cells and plotting dm/dt versus m. This should show clearly that growth is exponential on average.3) The authors show that the changes in dry mass and dry volume are very different frorm cell to cell. How does that relate to the extent of mitotic swelling, which is happening at the same time? Is there a correlation between the two: do cells that swell more also lose more dry mass and/or dry volume (or increase more their dry density)? Plotting one against the other might help understanding whether the two processes are coupled. The authors show that when cells are treated with EIPA, they do not swell but still show an increase in dry density. But it does not mean that the two processes are not coupled in control cells. EIPA treatment is changing the capacity of the cell to swell (for unclear reasons – what it does the most directly is to change the intracellular pH) and it could decouple the two processes but it does not mean that might not share a common driving mechanism (in addition to mitotic entry of course). So it might be an informative plot to make.4) At the bottom of page 10 the authors discuss inhibiting 'cell elongation', which is a characteristic of anaphase cells. But they also then inhibit the next step, called furrowing, which is an even more drastic change of shape (when the cell divides in two). They could also mention it as a change of shape that does not affect the dry mass density.5) For the readers who are interested in more details of these experiments, it might be interesting to give, in the sup figures, the distribution of volumes and dry mass of the cells which have been treated with the various drugs , as they are given only normalized in the main figure. It would be interesting to know whether the drugs are changing these values . This is more curiosity from this referee…so the authors might as well choose not to show all that.6) In the discussion, line 322, the authors mention high temporal resolution without phototoxicity as an advantage of their method over quantitative phase microscopy. QPM also allows high temporal resolution without phototoxicity. But the main point I think is that it does not measure the same thing! In particular, it does not give access to this 'dry volume' parameter.7) The results seem to be cell type dependent and it is quite surprising that the dry mass loss is different for L1210 expressing the FUCCI marker compared with wild-type L1210. Can the authors speculate on why this could be the case (e.g. different levels of exocytosis activity?). Quantification of other cell types in the main figure would be helpful. In line with this, the reduction in dry mass loss with Brefeldin A or Vacuolin-1 inhibition in Figure 6 is convincing but comparable to that observed in wild type cells, while the FUCCI cells show more dramatic decrease in cell mass. The authors should acknowledge this clearly in the text and soften the claim that chemical inhibition of lysosomal exocytosis prevents mitotic dry composition changes.8) 'cell elongation' (page 10): do cells elongate in suspension?9) Does the mitotic exocytosis described in this relate to previous observations that cells 'bleb' to help correct for asymmetric positioning of the mitotic spindle (see https://doi.org/10.1038/nature14496 for example?)10) The method seems to affect the dynamics of mitosis. Line 149 page 6 states 'while limited D2O exposure appears to influence mitosis duration, it does not radically …..' This caveat should be mentioned again in the discussion. Essential revisions:1) All the reviewers request that you clarify the definition of dry mass, dry density, dry mass density, and dry volume. Below you will see the comments of each reviewer on this point, which should help you to clarify these terms.Thank you for this feedback. We apologize for the confusion caused by our terminology, which was based on the terminology used in our original manuscript . We have now changed our terminology to be consistent with more recent works by others. We now define ‘dry density’ as dry mass divided by total volume, and we define our measurements of dry mass divided by dry volume as ‘dry mass density’. This change has been applied throughout our manuscript, including our manuscript title. In addition, we have added clearer definitions of each term to our Introduction and Measurement Method sections. Furthermore, we have minimized the use of the term ‘dry composition’ throughout our manuscript, as we now realize this may cause confusion to some readers.More specifically, our introduction (page 3) now states: “Here, we introduce a new approach for monitoring single cell’s dry mass , dry volume , and density of the dry mass (i.e. dry mass / dry volume), which we will refer to as dry mass density.” These definitions are also repeated in our Measurement Method section (page 4), as many readers may look for the definitions in that section.2) A mathematical model could help to clarify the interpretation of your results. Referee 3 suggests a simple mathematical model that would roughly estimate how the cell composition  should change and what the composition of the lost (secreted) components should be to provide the observed changes to the dry mass and density, given the existing information on average cell composition and the densities of different biomolecules .Thank you for this feedback. We fully agree that such calculations could be very useful in interpreting our results. We have now estimated how much lipids a theoretical cell would have to lose in order to explain the dry mass loss and dry mass density increase. This is explained in a new paragraph  as follows:“The magnitude of dry mass density increase in mitosis was large. We have previously observed similar magnitude changes in dry mass density when perturbing proliferation in mammalian cell . To provide some rough estimates of what kind of compositional changes would be required to achieve the dry mass loss and dry mass density increase, we carried out a back-of-the-envelope calculations. Assuming a typical mammalian cell composition and typical macromolecule dry mass densities , we calculated the degree of lipid loss needed to increase dry mass density by 2.5%. This suggested that cells would have to secrete ~1/3 of their lipid content in early mitosis. This could be achieved via lysosomal exocytosis of lipids. Lipid droplets, the main lipid storages inside cells, are frequently trafficked into and degraded in lysosomes , and lipid droplets can also be secreted via lysosomal exocytosis . However, it seems likely that the mitotic dry mass density increase also involves secretion of other low dry mass density components  and/or a minor, transient increase in high dry mass density components  in early mitosis. Indeed, CDK1 activity has been suggested to drive a transient increase in protein and RNA content in early mitosis .”3) Some of the drug treatments require more replicates to provide more conclusive answers. In particular, Figure 4E-F: Given the SD for barasertib and EIPA cells, the authors need more than ~6-10 cells to draw conclusions based on treatment effects. That barasertib treatment does not affect dry mass and dry density changes is not yet convincing.eLife editors, we have instead softened our conclusions to acknowledge that Barasertib treated cells could still have changes in their dry mass behavior when compared to control cells. Our updated Results section (page 9)Yes, we agree that the Barasertib treatment experiments would require more replicates in order to have full confidence that the drug does not influence dry mass and dry mass density behavior in mitosis. After consulting the “The mitotic changes in cell’s dry mass and dry mass density cannot be fully explained by cell elongation and furrowing, although cell elongation and furrowing (or Aurora B activity) could still have an influence on dry mass and dry mass density dynamics in mitosis.”We have also changed our figure 4 title to “Mitotic dry mass loss and dry mass density increase do not require morphological changes”, and we have removed the sentence from our abstract that claimed that dry mass changes are independent from cytokinesis.Other comments:1) The authors show that their measure does not depend on the time spent in D2O, which is important. But it means that the water needs only a few seconds to exchange. Is that expected given the permeation of water through the plasma membrane and the size of the cell? I did not make the calculation, but it seems a bit short. Can the authors give also the theoretical estimate based on the permeation of water through the membrane?Thank you for the feedback, this is a great suggestion. We have now carried out rough estimations of the water exchange time, as based on the water permeability of aquaporin channels, the typical abundance of aquaporin channels and the typical volume of a L1210 cell. We have also added more references that provide direct experimental support for the time required to exchange cellular water content. We detail these at the end of our Measurement method section (page 5) as follows:“Our measurement principle involves the cells exchanging their intracellular water content between H2O and D2O, and this exchange has to be complete for our measurements to reflect the dry mass and dry mass density of cells. Direct experimental evidence suggests that cells normally exchange their water content within ~1 second . To support this, we carried out back-of-the-envelope calculations on the intracellular water exchange times, as based on reported aquaporin water permeability , abundance  and a cell water volume of ~1000 fl (approximate water volume for a L1210 cell). This suggested that cells are capable of exchanging all of their water volume within 0.1 – 2.5 s, depending on the reported aquaporin water permeability. Notably, the true intracellular water exchange time may differ from this due to intracellular water diffusion, water molecule interactions with other biomolecules and additional means of water transport in and out of the cell .”2) For the measure of the exponential growth rate, the authors could use the same approach as in Cadart et al. Nat Com 2018, which might be more robust than fitting on the single cell growth curve: measuring the growth speed (dm/dt) on short periods of time all along the cell growth trajectories and for all cells and plotting dm/dt versus m. This should show clearly that growth is exponential on average.Thank you for the suggestion, we have now added the recommended plot, which clearly shows that growth rates (pg/h) increase as cell mass increases within L1210 cell interphases. This is detailed on pages 6 and 7 of our manuscript, stating that: “Consistent with exponential growth, instantaneous dry mass growth rates (pg/h) increased as a function of dry mass .”3) The authors show that the changes in dry mass and dry volume are very different frorm cell to cell. How does that relate to the extent of mitotic swelling, which is happening at the same time? Is there a correlation between the two: do cells that swell more also lose more dry mass and/or dry volume (or increase more their dry density)? Plotting one against the other might help understanding whether the two processes are coupled. The authors show that when cells are treated with EIPA, they do not swell but still show an increase in dry density. But it does not mean that the two processes are not coupled in control cells. EIPA treatment is changing the capacity of the cell to swell (for unclear reasons – what it does the most directly is to change the intracellular pH) and it could decouple the two processes but it does not mean that might not share a common driving mechanism (in addition to mitotic entry of course). So it might be an informative plot to make.Thank you for the feedback. Unfortunately, our measurement capabilities do not allow us to simultaneously measure the dry mass (and dry mass density) and the total volume of a cell, and developing such methods would require major engineering work. Thus, we cannot at the moment correlate the degree of mitotic cell swelling to the degree of mitotic dry mass loss. That being said, we agree with the reviewer’s comment, and we have now added the following conclusion to our manuscript :“The mitotic dry mass and dry mass density changes do not require mitotic cell swelling. However, it remains possible that these two phenotypes share a common driving mechanism.”4) At the bottom of page 10 the authors discuss inhibiting 'cell elongation', which is a characteristic of anaphase cells. But they also then inhibit the next step, called furrowing, which is an even more drastic change of shape (when the cell divides in two). They could also mention it as a change of shape that does not affect the dry mass density.Thank you for pointing this out, as we do indeed prevent furrowing using Aurora B inhibition. We have now pointed this out to the reader in the relevant Results section (page 9).5) For the readers who are interested in more details of these experiments, it might be interesting to give, in the sup figures, the distribution of volumes and dry mass of the cells which have been treated with the various drugs , as they are given only normalized in the main figure. It would be interesting to know whether the drugs are changing these values . This is more curiosity from this referee…so the authors might as well choose not to show all that.Thank you for the suggestion, but we have chosen not to add such figures. Cell dry mass and dry volume vary significantly between individual cells even when examining all cells specifically at mitotic entry, especially since the cells are measured over a timespan of many months. In the absence of significantly larger datasets, we find such examinations to have little value. The normalization we utilize in the figures allows us to examine the mitosis specific effects despite this inherent size variability. However, all our raw data is attached to our manuscript, so these analyses can be carried out by others who consider them more important.6) In the discussion, line 322, the authors mention high temporal resolution without phototoxicity as an advantage of their method over quantitative phase microscopy. QPM also allows high temporal resolution without phototoxicity. But the main point I think is that it does not measure the same thing! In particular, it does not give access to this 'dry volume' parameter.Thank you for pointing this out. We have indeed acknowledged in our discussion that our method can measure the dry volume of cells, which makes our method unique. We have also adjusted our text in the discussion to avoid claims that QPM could not achieve high temporal resolution.7) The results seem to be cell type dependent and it is quite surprising that the dry mass loss is different for L1210 expressing the FUCCI marker compared with wild-type L1210. Can the authors speculate on why this could be the case (e.g. different levels of exocytosis activity?). Quantification of other cell types in the main figure would be helpful. In line with this, the reduction in dry mass loss with Brefeldin A or Vacuolin-1 inhibition in Figure 6 is convincing but comparable to that observed in wild type cells, while the FUCCI cells show more dramatic decrease in cell mass. The authors should acknowledge this clearly in the text and soften the claim that chemical inhibition of lysosomal exocytosis prevents mitotic dry composition changes.Thank you for this feedback. The cell type-dependency of our results is indeed quite surprising, although we have previously also observed that mitotic energy metabolism can differ significantly even between the wt and FUCCI expressing L1210 cells . For all of these phenotypes, we have not found any solid evidence that would explain the cell type-dependency. It is possible that baseline levels of endo- and exocytosis could explain the difference in dry mass loss in mitosis, but in the absence of more evidence, we would prefer to avoid this discussion in our manuscript. However, we completely agree with the reviewer that our conclusions should be rephrased. Our updated Results section (page 11) now states that:“Inhibitors of lysosomal exocytosis decrease mitotic dry mass loss and dry mass density increase” (section title), and that “Lysosomal exocytosis is, at least partly, responsible for the loss of dry mass and increased dry mass density in early mitosis. However, other mechanisms, including other forms of exocytosis, may also be involved, because inhibition of lysosomal exocytosis did not prevent all dry mass loss in mitosis, and because our results are limited to a single cell line.”In addition, we have now stated in our discussion (pages 13) that:“Other mechanisms, especially other forms of exocytosis, may also be involved, as we were not able to completely block the dry mass loss in early mitosis by preventing lysosomal exocytosis.”8) 'cell elongation' (page 10): do cells elongate in suspension?Yes, even in suspension cells the phenotype observed during early anaphase corresponds to cell elongation. This is evident, at least to some degree, in our Figure 4B, where we display timelapse imaging of our suspension grown L1210 FUCCI cells (imaging is carried out in suspension culture). Yet, to avoid confusion, we now call this “cell elongation and furrowing”, as the detailed reviewer comments noted that cell furrowing is a distinct cytokinetic event that we also inhibit.9) Does the mitotic exocytosis described in this relate to previous observations that cells 'bleb' to help correct for asymmetric positioning of the mitotic spindle (see https://doi.org/10.1038/nature14496 for example?)This is an interesting hypothesis, but the timing of cell dry mass loss seems to precede the cell blebbing. More specifically, our data shows that cell mass loss takes place predominantly from mitotic entry (prophase) to metaphase-anaphase transition, whereas the polar relaxation of actin cortex and the consequent cell blebbing takes place predominantly in anaphase. Notably, we have examined polar relaxation in our L1210 FUCCI cells  and the timing of this phenotype is the same in our L1210 cells as in the other model systems examined in the literature . We therefore consider it unlikely that the cell blebbing typically observed in mitosis would be part of the mechanism causing dry mass loss. Yet, the mitotic exocytosis provides excess plasma membrane (as we note in our discussion), and this excess membrane could enable or even promote blebbing10) The method seems to affect the dynamics of mitosis. Line 149 page 6 states 'while limited D2O exposure appears to influence mitosis duration, it does not radically …..' This caveat should be mentioned again in the discussion.Thank you for pointing this out. In our updated Discussion section (on page 12), we now state that:“However, our measurement approach also decreased the duration of mitosis and cytokinesis, which could potentially influence the dry mass dynamics we observe in mitosis.”


Preadolescent male rats were injected with MOD  or a vehicle for 14 days . At postnatal day 36, we measured the GLU and GABA contents and their extracellular levels in the nucleus accumbens (NAc). In addition, the GLU and GABA contents were measured in the ventral tegmental area (VTA) and D2 protein levels in the prefrontal cortex (PFC). Chronic use of MOD during adolescence induces behavioral and neurochemical changes associated with the mesocorticolimbic system, such as a reduction in PFC D2 expression, VTA GABA levels and NSA. These results contribute to the understanding of the neurological effects of chronic MOD use on a young healthy brain.The misuse of psychostimulants is an increasing behavior among young people, highlighting in some countries the abuse of modafinil (MOD) as a neuropotentiator. However, several clinical trials are investigating MOD as an alternative pharmacological treatment for attentional deficit and hyperactivity disorder (ADHD) in children and adolescents. On the other hand, the early use of psychostimulants and the misdiagnosis rates in ADHD make it crucial to investigate the brain effects of this type of drug in young healthy individuals. The aim of this work was to evaluate the effects of chronic MOD treatment on neurochemicals (γ-aminobutyric acid and glutamate), dopamine receptor 2 (D Modafinil (MOD) is a wakefulness-promoting drug and an atypical psychostimulant that is commonly prescribed for narcolepsy, obstructive sleep apnea/hypopnea syndrome, and shift-work sleep disorder ,2. MOD iThe improvement of ADHD symptoms using psychostimulants is related to dopamine (DA) neurons within the mesocorticolimbic circuitry ,9,10. MOB) regulate the NAc and VTA DA release  mazindol and decrease D2 binding in the PFC 66]. Brie+ stimuli intra-NAc samples. The Kruskal–Wallis test followed by Dunn post-hoc was used to determine significant differences between groups in both time points (day 1 versus day 14) in the NSA and rearing frequency test. The significance was set at p < 0.05. The statistical analysis was carried out in GraphPad Prism v9.0 . One rat from the MOD group was an outlier (more than 2 times S.D. + mean) in the GABA tissue content level analysis and was removed from the analysis. One rat from the vehicle group was excluded due to zero rearing frequency at day 1.All data were analyzed with the D’Agostino–Pearson test to assess normality. The Mann–Whitney test with Welch’s correction was used to determine significant differences in GLU and GABA tissue content in the NAc and VTA. A one-way ANOVA followed by Tukey post-hoc tests was used to determine significant differences between basal micro dialysate samples and post-70 mM K2 expression in the PFC, and acute modification of NSA. These results contribute to the understanding of the neurophysiological and chemical mechanisms underlying the chronic use of MOD at critical periods of development, suggesting that prolonged use of MOD induces tolerance, modifying both behavioral and neurochemical substrates from the first day of use compared to the vehicle. Finally, it is important to continue investigating the short- and long-term effects on the brain reward circuitry after chronic MOD administration. This drug is gaining popularity among young students, and ADHD is highly misdiagnosed in children and adolescents, which is a sensitive population to be exposed to psychostimulants.Our results show that the chronic use of MOD during a critical development period induces behavioral and neurochemical changes in the mesocorticolimbic circuitry, specifically altering GABA levels in the VTA, D