In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.

Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.

Fission Yeast TORC2 Signaling Pathway Ensures Cell Proliferation under Glucose-Limited, Nitrogen-Replete Conditions.

Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast, Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.

TORC2 inhibition of α-arrestin Aly3 mediates cell surface persistence of S. pombe Ght5 glucose transporter in low glucose.

In the fission yeast, Schizosaccharomyces pombe, the high-affinity hexose transporter, Ght5, must be transcriptionally upregulated and localized to the cell surface for cell division under limited glucose. Although cell-surface localization of Ght5 depends on Target of rapamycin complex 2 (TORC2), the molecular mechanisms by which TORC2 ensures proper localization of Ght5 remain unknown. We performed genetic screening for gene mutations that restore Ght5 localization on the cell surface in TORC2-deficient mutant cells, and identified a gene encoding an uncharacterized α-arrestin-like protein, Aly3/SPCC584.15c. α-arrestins are thought to recruit a ubiquitin ligase to membrane-associated proteins. Consistently, Ght5 is ubiquitylated in TORC2-deficient cells, and this ubiquitylation is dependent on Aly3. TORC2 supposedly enables cell-surface localization of Ght5 by preventing Aly3-dependent ubiquitylation and subsequent ubiquitylation-dependent translocation of Ght5 to vacuoles. Surprisingly, nitrogen starvation, but not glucose depletion, triggers Aly3-dependent transport of Ght5 to vacuoles in S. pombe, unlike budding yeast hexose transporters, vacuolar transport of which is initiated upon changes in hexose concentration. This study provides new insights into the molecular mechanisms controlling the subcellular localization of hexose transporters in response to extracellular stimuli.

Multiple nutritional phenotypes of fission yeast mutants defective in genes encoding essential mitochondrial proteins.

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe, 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive (ts) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and 'super-housekeeping' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.

Publisher Correction: Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding.

Change history: In Fig. 1b and c of this Letter, the inset times in the DIC and GFP microscopy images should be in minutes ('min') instead of seconds ('s'). This has not been corrected online.

Extracellular glucose level regulates dependence on GRP78 for cell surface localization of multipass transmembrane proteins in HeLa cells.

Many human-cultured cell lines survive glucose starvation, but the underlying mechanisms remain unclear. Here, we searched for proteins required for cellular adaptation to glucose-limited conditions and identified several endoplasmic reticulum chaperones in the glucose-regulated protein (GRP) family as proteins enriched in the cellular membrane. Surprisingly, these proteins, which are required for cell surface localization of GLUT1 under high-glucose conditions, become dispensable for targeting GLUT1 to the surface upon glucose starvation. In marked contrast, cell surface localization of single-pass transmembrane proteins, such as epidermal growth factor receptor and CD98, is not disturbed by GRP78 depletion regardless of the extracellular glucose level. These results indicate that the extracellular glucose level regulates dependence on the GRPs for cell surface localization of multipass transmembrane proteins.

Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding.

To divide, most animal cells drastically change shape and round up against extracellular confinement. Mitotic cells facilitate this process by generating intracellular pressure, which the contractile actomyosin cortex directs into shape. Here, we introduce a genome-scale microcantilever- and RNAi-based approach to phenotype the contribution of > 1000 genes to the rounding of single mitotic cells against confinement. Our screen analyzes the rounding force, pressure and volume of mitotic cells and localizes selected proteins. We identify 49 genes relevant for mitotic rounding, a large portion of which have not previously been linked to mitosis or cell mechanics. Among these, depleting the endoplasmic reticulum-localized protein FAM134A impairs mitotic progression by affecting metaphase plate alignment and pressure generation by delocalizing cortical myosin II. Furthermore, silencing the DJ-1 gene uncovers a link between mitochondria-associated Parkinson's disease and mitotic pressure. We conclude that mechanical phenotyping is a powerful approach to study the mechanisms governing cell shape.

Rheology of the Active Cell Cortex in Mitosis.

The cell cortex is a key structure for the regulation of cell shape and tissue organization. To reach a better understanding of the mechanics and dynamics of the cortex, we study here HeLa cells in mitosis as a simple model system. In our assay, single rounded cells are dynamically compressed between two parallel plates. Our measurements indicate that the cortical layer is the dominant mechanical element in mitosis as opposed to the cytoplasmic interior. To characterize the time-dependent rheological response, we extract a complex elastic modulus that characterizes the resistance of the cortex against area dilation. In this way, we present a rheological characterization of the cortical actomyosin network in the linear regime. Furthermore, we investigate the influence of actin cross linkers and the impact of active prestress on rheological behavior. Notably, we find that cell mechanics values in mitosis are captured by a simple rheological model characterized by a single timescale on the order of 10 s, which marks the onset of fluidity in the system.

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement.

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

A human interactome in three quantitative dimensions organized by stoichiometries and abundances.

The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.

Adaptive regulation of glucose transport, glycolysis and respiration for cell proliferation.

The cell must utilise nutrients to generate energy as a means of sustaining its life. As the environment is not necessarily abundant in nutrients and oxygen, the cell must be able to regulate energy metabolism to adapt to changes in extracellular and intracellular conditions. Recently, several key regulators of energy metabolism have been reported. This review describes the recent advances in molecular regulation of energy metabolism, focusing mainly on glycolysis and its shunt pathways. Human diseases, such as cancer and neurodegenerative disorders, are also discussed in relation to failure of energy metabolism regulation.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

Products of the Parkinson's disease-related glyoxalase DJ-1, D-lactate and glycolate, support mitochondrial membrane potential and neuronal survival.

Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes linked with the onset of Parkinson's disease, DJ-1/PARK7, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease.

C11ORF24 is a novel type I membrane protein that cycles between the Golgi apparatus and the plasma membrane in Rab6-positive vesicles.

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

A conserved RhoGAP limits M phase contractility and coordinates with microtubule asters to confine RhoA during cytokinesis.

During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis and cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M phase, leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions.

A genomic toolkit to investigate kinesin and myosin motor function in cells.

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

APC15 mediates CDC20 autoubiquitylation by APC/C(MCC) and disassembly of the mitotic checkpoint complex.

The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/C(CDC20)) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/C(MCC))-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/C(CDC20) and APC/C(CDH1). Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/C(CDC20) and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.

Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp.

To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance force, mechanical stress and volume of globular animal cells under compression. We describe the AFM and cantilever setup, live cell culture in the AFM, how to ensure stability of AFM measurements during medium perfusion, integration of optical microscopy to measure parameters such as volume and track intracellular dynamics, and interpretation of the physical parameters measured. Although we use this protocol on trypsinized interphase and mitotic HeLa cells, it can also be applied to other cells with a relatively globular shape, especially animal cells in a low-adhesive environment. After a short setup phase, the protocol can be used to investigate approximately one cell per hour.

Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation.

Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1-encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.

Force probing cell shape changes to molecular resolution.

Atomic force microscopy (AFM) is a force sensing nanoscopic tool that can be used to undertake a multiscale approach to understand the mechanisms that underlie cell shape change, ranging from the cellular to molecular scale. In this review paper, we discuss the use of AFM to characterize the dramatic shape changes of mitotic cells. AFM-based mechanical assays can be applied to measure the considerable rounding force and hydrostatic pressure generated by mitotic cells. A complementary AFM technique, single-molecule force spectroscopy, is able to quantify the interactions and mechanisms that functionally regulate individual proteins. Future developments of these nanomechanical methods, together with advances in light microscopy imaging and cell biological and genetic tools, should provide further insight into the biochemical, cellular and mechanical processes that govern mitosis and other cell shape change phenomena.