Search

Understanding protein function is pivotal in comprehending the intricate mechanisms that underlie many crucial biological activities, with far-reaching implications in the fields of medicine, biotechnology, and drug development. However, more than 200 million proteins remain uncharacterized, and computational efforts heavily rely on protein structural information to predict annotations of varying quality. Here, we present a method that utilizes statistics-informed graph networks to predict protein functions solely from its sequence. Our method inherently characterizes evolutionary signatures, allowing for a quantitative assessment of the significance of residues that carry out specific functions. PhiGnet not only demonstrates superior performance compared to alternative approaches but also narrows the sequence-function gap, even in the absence of structural information. Our findings indicate that applying deep learning to evolutionary data can highlight functional sites at the residue level, providing valuable support for interpreting both existing properties and new functionalities of proteins in research and biomedicine., (© 2024. The Author(s).)

Lipid trafficking is critical for the biogenesis and expansion of organelle membranes. Lipid transport proteins (LTPs) have been proposed to facilitate lipid transport at contact sites between organelles. Despite the fundamental importance of LTPs in cell physiology, our knowledge on the mechanisms of interorganelle lipid distribution remains poor due to the scarcity of assays to monitor lipid flux in vivo. In this review, we highlight the recent development of a versatile method named METALIC (Mass tagging-Enabled Tracking of Lipids in Cells), which uses a combination of enzymatic mass tagging and mass spectrometry to track lipid flux between organelles inside living cells. We discuss the methodology, its distinct advantages, limitations as well as its potential to unearth the pipelines of lipid transport and LTP function in vivo., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact role of Miro in these functions is unclear but involves binding to a variety of client proteins. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro interactors-namely, CENPF, Trak, and MYO19-all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the first calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including MTFR1/2/1L, the lipid transporters Mdm34 and VPS13D, and the ubiquitin E3-ligase Parkin. Given the shared binding mechanism of these functionally diverse clients and its conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health., (© 2024. The Author(s).)

The non-vesicular transport of lipids between organelles mediated by lipid transport proteins (LTPs) is a key determinant of organelle biogenesis and function. Despite performing a vital function in organelle homeostasis, none of the LTP-encoding genes identified so far are truly essential, even in the simple genome of yeast, suggesting widespread redundancy. In line with this fact, it has been found that a number of LTPs have overlapping functions, making it challenging to assign unique roles for an individual LTP in lipid distribution. In our genetic screens under stringent conditions in which the distinct function of an LTP might become essential, we stumbled upon Csf1, a highly conserved protein with a Chorein-N motif found in other lipid transporters and unraveled a new function for Csf1 in lipid remodeling and homeoviscous adaptation of the lipidome. Here, we further speculate on the potential mechanisms of how the putative function of Csf1 in lipid transport could be intimately connected to its role in lipid remodeling across organelles., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

The distinct activities of organelles depend on the proper function of their membranes. Coordinated membrane biogenesis of different organelles necessitates lipid transport from their site of synthesis to their destination. Several factors have been proposed to participate in lipid distribution, but despite its basic importance, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. A reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside cells. In this strategy, two enzymes, one directed to a 'donor' and the other to an 'acceptor' organelle, add two distinct mass tags to lipids. Mass-spectrometry-based detection of lipids bearing the two mass tags is then used to quantify exchange between the two organelles. By applying this approach, we show that the ERMES and Vps13-Mcp1 complexes have transport activity in vivo, and unravel their relative contributions to endoplasmic reticulum-mitochondria lipid exchange., (© 2022. The Author(s).)

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non-vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1-an uncharacterized protein harboring a Chorein-N lipid transport motif-for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. The technology combines atomic force microscopy, optical microscopy, and nanofluidics to achieve force and volume control with real-time inspection. We developed dedicated probes that allow minimally invasive entry into cells and optimized fluid flow to extract specific organelles. When extracting single or a defined number of mitochondria, their morphology transforms into a pearls-on-a-string phenotype due to locally applied fluidic forces. We show that the induced transition is calcium independent and results in isolated, intact mitochondria. Upon cell-to-cell transplantation, the transferred mitochondria fuse to the host cells mitochondrial network. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed monitoring of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 minutes after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, donor mitochondrial DNA (mtDNA) was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for therapy, mechanobiology, and synthetic biology., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: One of the coauthors (E.S.) oversees the production of FluidFM tips and is employed by SmartTip (NL).

Genome-wide transposon mutagenesis followed by deep sequencing allows the genome-wide mapping of growth-affecting loci in a straightforward and time-efficient way.SAturated Transposon Analysis in Yeast (SATAY) takes advantage of a modified maize transposon that is highly mobilizable in S. cerevisiae. SATAY allows not only the genome-wide mapping of genes required for growth in select conditions (such as genetic interactions or drug sensitivity/resistance), but also of protein sub-domains, as well as the creation of gain- and separation-of-function alleles. From strain preparation to the mapping of sequencing reads, we detail all the steps for the making and analysis of SATAY libraries in any S. cerevisiae lab, requiring only ordinary equipment and access to a Next-Gen sequencing platform., (© 2022. The Author(s).)

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The development of the lens in the vertebrate eye requires the degradation of all organelles. In a recent issue of Nature, Morishita et al. (2021) identify a conserved phospholipase that appears to achieve this by simply digesting organelle membranes away., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Information on the co-evolution of amino acid pairs in a protein can be used for endeavors such as protein engineering, mutation design, and structure prediction. Here we report a method that captures significant determinants of proteins using estimated co-evolution information to identify networks of residues, termed "residue communities", relevant to protein function. On the benchmark dataset (67 proteins with both catalytic and allosteric residues), the Pearson's correlation between the identified residues in the communities at functional sites is 0.53, and it is higher than 0.8 by taking account of conserved residues derived from the method. On the endoplasmic reticulum-mitochondria encounter structure complex, the results indicate three distinguishable residue communities that are relevant to functional roles in the protein family, suggesting that the residue communities could be general evolutionary signatures in proteins. Based on the method, we provide a webserver for the scientific community to explore the signatures in protein families, which establishes a powerful tool to analyze residue-level profiling for the discovery of functional sites and biological pathway identification. This web-server is freely available for non-commercial users at https://kornmann.bioch.ox.ac.uk/leri/services/ecs.html, neither login nor e-mail required., Competing Interests: Potential conflicts of interest. N.J.C. (Y. Z.) is a founder of Leri Ltd based in Oxford, UK. All other authors report no conflicts of interest relevant to this article., (© 2021 The Authors.)

Indole-3-acetic acid (IAA) is the most common, naturally occurring phytohormone that regulates cell division, differentiation, and senescence in plants. The capacity to synthesize IAA is also widespread among plant-associated bacterial and fungal species, which may use IAA as an effector molecule to define their relationships with plants or to coordinate their physiological behavior through cell-cell communication. Fungi, including many species that do not entertain a plant-associated life style, are also able to synthesize IAA, but the physiological role of IAA in these fungi has largely remained enigmatic. Interestingly, in this context, growth of the budding yeast Saccharomyces cerevisiae is sensitive to extracellular IAA. Here, we use a combination of various genetic approaches including chemical-genetic profiling, SAturated Transposon Analysis in Yeast (SATAY), and genetic epistasis analyses to identify the mode-of-action by which IAA inhibits growth in yeast. Surprisingly, these analyses pinpointed the target of rapamycin complex 1 (TORC1), a central regulator of eukaryotic cell growth, as the major growth-limiting target of IAA. Our biochemical analyses further demonstrate that IAA inhibits TORC1 both in vivo and in vitro. Intriguingly, we also show that yeast cells are able to synthesize IAA and specifically accumulate IAA upon entry into stationary phase. Our data therefore suggest that IAA contributes to proper entry of yeast cells into a quiescent state by acting as a metabolic inhibitor of TORC1., Competing Interests: The authors have declared that no competing interests exist.

Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid overabundance stress. How cells form MDCs is unclear. Here, we show that MDCs are dynamic structures that form and stably persist at sites of contact between the ER and mitochondria. MDC biogenesis requires the ER-mitochondria encounter structure (ERMES) and the conserved GTPase Gem1, factors previously implicated in lipid exchange and membrane tethering at ER-mitochondria contacts. Interestingly, common genetic suppressors of abnormalities displayed by ERMES mutants exhibit distinct abilities to rescue MDC formation in ERMES-depleted strains and are incapable of rescuing MDC formation in cells lacking Gem1. Thus, the function of ERMES and Gem1 in MDC biogenesis may extend beyond their conventional role in maintaining mitochondrial phospholipid homeostasis. Overall, this study identifies an important function for ER-mitochondria contacts in the biogenesis of MDCs., (© 2020 English et al.)

Eukaryotic cells are compartmentalized into organelles by intracellular membranes. While the organelles are distinct, many of them make intimate contact with one another. These contacts were first observed in the 1950s, but only recently have the functions of these contact sites begun to be understood. In yeast, the endoplasmic reticulum (ER) makes extensive intermembrane contacts with the plasma membrane (PM), covering ∼40% of the PM. Many functions of ER-PM contacts have been proposed, including nonvesicular lipid trafficking, ion transfer, and as signaling hubs. Surprisingly, cells that lack ER-PM contacts grow well, indicating that alternative pathways may be compensating for the loss of ER-PM contact. To better understand the function of ER-PM contact sites we used saturating transposon mutagenesis to identify synthetic lethal mutants in a yeast strain lacking ER-PM contact sites. The strongest hits were components of the ESCRT complexes. The synthetic lethal mutants have low levels of some lipid species but accumulate free fatty acids and lipid droplets. We found that only ESCRT-III components are synthetic lethal, indicating that Vps4 and other ESCRT complexes do not function in this pathway. These data suggest that ESCRT-III proteins and ER-PM contact sites act in independent pathways to maintain lipid homeostasis.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1 wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S phase-dependent manner to assist in the eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among the potential Ddi1 targets, we found the core component of Pol II and show that its genotoxin-induced degradation is impaired in ddi1. We propose that the Ddi1 protease contributes to DPC proteolysis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hereditary sensory and autonomic neuropathy (HSAN) types IA and IC (IA/C) are caused by elevated levels of an atypical class of lipid named 1-deoxysphingolipid (DoxSL). How elevated levels of DoxSL perturb the physiology of the cell and how the perturbations lead to HSAN IA/C are largely unknown. In this study, we show that C 26 -1-deoxydihydroceramide (C 26 -DoxDHCer) is highly toxic to the cell, while C 16 - and C 18 -DoxDHCer are less toxic. Genome-wide genetic screens and lipidomics revealed the dynamics of DoxSL accumulation and DoxSL species responsible for the toxicity over the course of DoxSL accumulation. Moreover, we show that disruption of F-actin organization, alteration of mitochondrial shape, and accumulation of hydrophobic bodies by DoxSL are not sufficient to cause complete cellular failure. We found that cell death coincides with collapsed ER membrane, although we cannot rule out other possible causes of cell death. Thus, we have unraveled key principles of DoxSL cytotoxicity that may help to explain the clinical features of HSAN IA/C.

Mechanobiology studies from the last decades have brought significant insights into many domains of biological research, from development to cellular signaling. However, mechano-regulation of subcellular components, especially membranous organelles, are only beginning to be unraveled. In this paper, we take mitochondrial mechanobiology as an example to discuss recent advances and current technical challenges in this field. In addition, we discuss the needs for future toolbox development for mechanobiological research of intracellular organelles.

Over the last years, the importance of inter-organelle communication has become more and more evident, attested by the fast growing number of newly-identified membrane contact sites (MCS). At MCSs two organelles are connected via protein tethers that bring them in close proximity to facilitate metabolite exchange. In this review, we will focus on the MCSs connecting the ER and mitochondria, which have been implicated in phospholipid transport. While we already know the molecular identity of some tethers, we are still far from understanding the mechanisms underlying the phospholipid transport processes. In vitro studies suggest that some proteins in MCSs are capable of transporting lipids, however only at rates that do not meet the mitochondrial lipid demand. In vivo studies are even more puzzling as it appears that many redundant lipid transport routes, involving various lipid transport proteins and various MCSs, compensate for each other when necessary. Here, we will discuss the challenges in interpreting the data on lipid transport between ER and mitochondria from in vitro and in vivo experiments by highlighting some critical aspects that might be worth addressing in the future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.

CENP-F is a large, microtubule-binding protein that regulates multiple cellular processes including chromosome segregation and mitochondrial trafficking at cytokinesis. This multiplicity of functions is mediated through the binding of various partners, like Bub1 at the kinetochore and Miro at mitochondria. Due to the multifunctionality of CENP-F, the cellular phenotypes observed upon its depletion are difficult to interpret and there is a need to genetically separate its different functions by preventing binding to selected partners. Here we engineer a CENP-F point-mutant that is deficient in Miro binding and thus is unable to localize to mitochondria, but retains other localizations. We introduce this mutation in cultured human cells using CRISPR/Cas9 system and show it causes a defect in mitochondrial spreading similar to that observed upon Miro depletion. We further create a mouse model carrying this CENP-F variant, as well as truncated CENP-F mutants lacking the farnesylated C-terminus of the protein. Importantly, one of these truncations leads to ~80% downregulation of CENP-F expression. We observe that, despite the phenotypes apparent in cultured cells, mutant mice develop normally. Taken together, these mice will serve as important models to study CENP-F biology at organismal level. In addition, because truncations of CENP-F in humans cause a lethal disease termed Strømme syndrome, they might also be relevant disease models., Competing Interests: The authors have declared that no competing interests exist.

The intracellular environment of eukaryotic cells is highly complex and compact. The limited volume of the cell, usually a few hundred femtoliters, is not only occupied by numerous complicated, diverse membranous and proteinaceous structures, these structures are also highly dynamic due to constant remodeling and trafficking events. Consequently, intracellular interactions are more than just opportunities to exchange molecules; they also involve components physically navigating around each other in a highly confined space. While the biochemical interactions between organelles have been intensely studied in the past decades, the mechanical properties of organelles and the physical interactions between them are only beginning to be unraveled. Indeed, recent studies show that intracellular organelles are, at times, under extreme mechanical strain both in widely used experimental systems as well as in vivo In this Hypothesis, we highlight known examples of intracellular mechanical challenges in biological systems and focus on the coping mechanisms of two important organelles, the nucleus and mitochondria, for they are the best studied in this aspect. In the case of mitochondria, we propose that ER-mitochondrial contact sites at thin cell peripheries may induce mitochondrial fission by mechanically constricting mitochondrial tubules. We also briefly discuss the mechano-responsiveness of other organelles and interesting directions for future research., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes., (© 2018 Kawano et al.)

Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria - via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces - results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm.

Membrane contact sites between endoplasmic reticulum (ER) and mitochondria, mediated by the ER-mitochondria encounter structure (ERMES) complex, are critical for mitochondrial homeostasis and cell growth. Defects in ERMES can, however, be bypassed by point mutations in the endosomal protein Vps13 or by overexpression of the mitochondrial protein Mcp1. How this bypass operates remains unclear. Here we show that the mitochondrial outer membrane protein Mcp1 functions in the same pathway as Vps13 by recruiting it to mitochondria and promoting its association to vacuole-mitochondria contacts. Our findings support a model in which Mcp1 and Vps13 work as functional effectors of vacuole-mitochondria contact sites, while tethering is mediated by other factors, including Vps39. Tethered and functionally active vacuole-mitochondria interfaces then compensate for the loss of ERMES-mediated ER-mitochondria contact sites., (© 2017 John Peter et al.)

Certain cationic peptides interact with biological membranes. These often-complex interactions can result in peptide targeting to the membrane, or in membrane permeation, rupture, and cell lysis. We investigated the relationship between the structural features of membrane-active peptides and these effects, to better understand these processes. To this end, we employed a computational method for morphing a membranolytic antimicrobial peptide into a nonmembranolytic mitochondrial targeting peptide by "directed simulated evolution." The results obtained demonstrate that superficially subtle sequence modifications can strongly affect the peptides' membranolytic and membrane-targeting abilities. Spectroscopic and computational analyses suggest that N- and C-terminal structural flexibility plays a crucial role in determining the mode of peptide-membrane interaction.

Dynamic microtubule ends exert pulling and pushing forces on intracellular membranes and organelles. However, the mechanical linkage of microtubule tips to their cargoes is poorly understood. CENP-F is a nonmotor microtubule-binding protein that participates in microtubule binding at kinetochores and in the mitotic redistribution of the mitochondrial network. CENP-F-driven mitochondrial transport is linked to growing microtubule tips, but the underlying molecular mechanisms are unknown. Here we show that CENP-F tracks growing microtubule ends in living cells. In vitro reconstitution demonstrates that microtubule tips can transport mitochondria and CENP-F-coated artificial cargoes over micrometer-long distances during both growing and shrinking phases. Based on these and previous observations, we suggest that CENP-F might act as a transporter of mitochondria and other cellular cargoes by attaching them to dynamic microtubule ends during both polymerization and depolymerization of tubulin., (© 2017 Kanfer et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput.

To fuel the tremendously fast replication of Plasmodium liver stage parasites, the endoplasmic reticulum (ER) must play a critical role as a major site of protein and lipid biosynthesis. In this study, we analysed the parasite's ER morphology and function. Previous studies exploring the parasite ER have mainly focused on the blood stage. Visualizing the Plasmodium berghei ER during liver stage development, we found that the ER forms an interconnected network throughout the parasite with perinuclear and peripheral localizations. Surprisingly, we observed that the ER additionally generates huge accumulations. Using stimulated emission depletion microscopy and serial block-face scanning electron microscopy, we defined ER accumulations as intricate dense networks of ER tubules. We provide evidence that these accumulations are functional subdivisions of the parasite ER, presumably generated in response to elevated demands of the parasite, potentially consistent with ER stress. Compared to higher eukaryotes, Plasmodium parasites have a fundamentally reduced unfolded protein response machinery for reacting to ER stress. Accordingly, parasite development is greatly impaired when ER stress is applied. As parasites appear to be more sensitive to ER stress than are host cells, induction of ER stress could potentially be used for interference with parasite development., (© 2016 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd.)

During mitosis, cells undergo massive deformation and reorganization, impacting on all cellular structures. Mitochondria, in particular, are highly dynamic organelles, which constantly undergo events of fission, fusion and cytoskeleton-based transport. This plasticity ensures the proper distribution of the metabolism, and the proper inheritance of functional organelles. During cell cycle, mitochondria undergo dramatic changes in distribution. In this review, we focus on the dynamic events that target mitochondria during mitosis. We describe how the cell-cycle-dependent microtubule-associated protein centromeric protein F (Cenp-F) is recruited to mitochondria by the mitochondrial Rho GTPase (Miro) to promote mitochondrial transport and re-distribution following cell division., (© 2016 Authors; published by Portland Press Limited.)

The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex tethers the endoplasmic reticulum and the mitochondria. It is thought to facilitate interorganelle lipid exchange and influence mitochondrial dynamics and mitochondrial DNA maintenance. Despite this important role, ERMES is not found in metazoans. Here, we identified single amino acid substitutions in Vps13 (vacuolar protein sorting 13), a large universally conserved eukaryotic protein, which suppress all measured phenotypic consequences of ERMES deficiency. Combined loss of VPS13 and ERMES is lethal, indicating that Vps13 and ERMES function in redundant pathways. Vps13 dynamically localizes to vacuole-mitochondria and to vacuole-nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition-regulated organelle network., (© 2015 Lang et al.)

Although chromosome partitioning during mitosis is well studied, the molecular mechanisms that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. Here we show that mitochondria interact with growing microtubule tips and are transported towards the daughter cell periphery at the end of mitosis. This phenomenon is promoted by the direct and cell cycle-dependent interaction of the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F. Cenp-F is recruited to mitochondria by Miro at the time of cytokinesis and associates with microtubule growing tips. Cells devoid of Cenp-F or Miro show decreased spreading of the mitochondrial network as well as cytokinesis-specific defects in mitochondrial transport towards the cell periphery. Thus, Miro and Cenp-F promote anterograde mitochondrial movement and proper mitochondrial distribution in daughter cells.

A standout feature of eukaryotic cells is the presence of organelles with distinct chemical compositions and physical properties, which aid in the accomplishment of specialized metabolic tasks. This complex topology, however, makes a permanent crosstalk between the organelles a necessity for the coordination of cellular function. While molecule exchange between organelles via the vesicular transport system has been extensively studied, communication via direct connections has only recently become a new matter of interest. These direct connections termed membrane contact sites (MCSs) represent zones of close proximity (10-30nm) between two organelles. Research in the past years has revealed a number of MCSs especially between the ER and almost every other organelle [1(•)]. In particular, the MCSs between the ER and the mitochondria have undergone intense investigation. While the quest for ER-mitochondria MCS components in human cells has led to the revelation of an ever growing number of potential factors, studies in the simpler eukaryote Saccharomyces cerevisiae revealed the actual existence of a molecular tether between the two organelles [2(••)]., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Repairing or disposing of a malfunctioning object is an everyday dilemma. Replacing an item may be quicker than repairing it, but may also be more costly. Cells are faced with the same options when their organelles are challenged. Ensuring the health of the mitochondrial network is of utmost importance for cellular health and, not surprisingly, mitochondrial quality control can take both the repair and disposal routes. Spectacular advances have been made in recent years and a picture is starting to emerge of what drives a cell to take one or the other path. Interestingly, mitochondrial quality control seems to be deficient in various medically relevant conditions, such as neurodegeneration and aging.

Membrane-bound organelles are a wonderful evolutionary acquisition of the eukaryotic cell, allowing the segregation of sometimes incompatible biochemical reactions into specific compartments with tailored microenvironments. On the flip side, these isolating membranes that crowd the interior of the cell, constitute a hindrance to the diffusion of metabolites and information to all corners of the cell. To ensure coordination of cellular activities, cells use a network of contact sites between the membranes of different organelles. These membrane contact sites (MCSs) are domains where two membranes come to close proximity, typically less than 30nm. Such contacts create microdomains that favor exchange between two organelles. MCSs are established and maintained in durable or transient states by tethering structures, which keep the two membranes in proximity, but fusion between the membranes does not take place. Since the endoplasmic reticulum (ER) is the most extensive cellular membrane network, it is thus not surprising to find the ER involved in most MCSs within the cell. The ER contacts diverse compartments such as mitochondria, lysosomes, lipid droplets, the Golgi apparatus, endosomes and the plasma membrane. In this review, we will focus on the common organizing principles underlying the many MCSs found between the ER and virtually all compartments of the cell, and on how the ER establishes a network of MCSs for the trafficking of vital metabolites and information. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A long-observed but often neglected property of cellular organelles is their ability to associate into junctions. Aspects of cell physiology appear more and more to depend upon these contact sites, as their central molecular components are being identified. Contact sites between the endoplasmic reticulum (ER) and the mitochondria are emerging as a prime example of such contacts. The physiological role of these contact sites, first thought to be limited to the facilitation of lipid and calcium exchange between the two organelles, is found to extend to unexpected aspects of mitochondria and ER functions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cellular organelles need to communicate in order to co-ordinate homoeostasis of the compartmentalized eukaryotic cell. Such communication involves the formation of membrane contact sites between adjacent organelles, allowing privileged exchange of metabolites and information. Using a synthetic protein designed to artificially tether the ER (endoplasmic reticulum) to mitochondria, we have discovered a yeast protein complex naturally involved in establishing and maintaining contact sites between these two organelles. This protein complex is physiologically involved in a plethora of mitochondrial processes, suggesting that ER-mitochondria connections play a central co-ordinating role in the regulation of mitochondrial biology. Recent biochemical characterization of this protein complex led to the discovery that GTPases of the Miro family are part of ER-mitochondria connections. The yeast Miro GTPase Gem1 localizes to ER-mitochondria interface and influences the size and distribution of mitochondria. Thus Miro GTPases may serve as regulators of the ER-mitochondria connection.