A Highlights from MBoC Selection: DLC-1 facilitates germ granule assembly in Caenorhabditis elegans embryo

Germ granules are cytoplasmic assemblies of RNA-binding proteins (RBPs) required for germ cell development and fertility. During the first four cell divisions of the Caenorhabditis elegans zygote, regulated assembly of germ (P) granules leads to their selective segregation to the future germ cell. Here we investigate the role of DLC-1, a hub protein implicated in stabilization and function of diverse protein complexes, in maintaining P granule integrity. We find that DLC-1 directly interacts with several core P granule proteins, predominantly during embryogenesis. The loss of dlc-1 disrupts assembly of P granule components into phase-separated organelles in the embryos, regardless of whether or not DLC-1 directly interacts with these proteins. Finally, we infer that P granule dispersal in the absence of dlc-1 is likely independent of DLC-1’s function as a subunit of the dynein motor and does not result from a loss of cell polarity.     

A Highlights from MBoC Selection: EHBP-1 Functions with RAB-10 during Endocytic Recycling in Caenorhabditis elegans

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.     

A Highlights from MBoC Selection: Mitochondrial Fragmentation Leads to Intracellular Acidification in Caenorhabditis elegans and Mammalian Cells

Mitochondrial structural dynamics are regulated through the opposing processes of membrane fission and fusion, which are conserved from yeast to man. The chronic inhibition of mitochondrial fusion as a result of genetic mutation is the cause of human autosomal dominant optic atrophy (ADOA) and Charcot-Marie-Tooth syndrome type 2A (CMT-2A). Here, we demonstrate that genetic fragmentation of the mitochondrial network in Caenorhabditis elegans induces cellular acidification in a broad range of tissues from the intestine, to body wall muscles, and neurons. Genetic epistasis analyses demonstrate that fragmentation itself, and not the loss of a particular protein, leads to acidosis, and the worm''s fitness matches the extent of acidification. We suggest that fragmentation may cause acidification through two distinct processes: oxidative signaling after the loss of the ability of the mitochondrial inner membrane to undergo fusion and lactic acidosis after the loss of outer membrane fusion. Finally, experiments in cultured mammalian cells demonstrate a conserved link between mitochondrial morphology and cell pH homeostasis. Taken together these data reveal a potential role for acidosis in the differing etiology of diseases associated with mitochondrial morphology defects such as ADOA and CMT-2A.     

A Highlights from MBoC Selection: A novel function for the Caenorhabditis elegans torsin OOC-5 in nucleoporin localization and nuclear import

Torsin proteins are AAA+ ATPases that localize to the endoplasmic reticular/nuclear envelope (ER/NE) lumen. A mutation that markedly impairs torsinA function causes the CNS disorder DYT1 dystonia. Abnormalities of NE membranes have been linked to torsinA loss of function and the pathogenesis of DYT1 dystonia, leading us to investigate the role of the Caenorhabditis elegans torsinA homologue OOC-5 at the NE. We report a novel role for torsin in nuclear pore biology. In ooc-5–mutant germ cell nuclei, nucleoporins (Nups) were mislocalized in large plaques beginning at meiotic entry and persisted throughout meiosis. Moreover, the KASH protein ZYG-12 was mislocalized in ooc-5 gonads. Nups were mislocalized in adult intestinal nuclei and in embryos from mutant mothers. EM analysis revealed vesicle-like structures in the perinuclear space of intestinal and germ cell nuclei, similar to defects reported in torsin-mutant flies and mice. Consistent with a functional disruption of Nups, ooc-5–mutant embryos displayed impaired nuclear import kinetics, although the nuclear pore-size exclusion barrier was maintained. Our data are the first to demonstrate a requirement for a torsin for normal Nup localization and function and suggest that these functions are likely conserved.     

A Highlights from MBoC Selection: Analyzing the Effects of Delaying Aster Separation on Furrow Formation during Cytokinesis in the Caenorhabditis elegans Embryo

Signaling by the centrosomal asters and spindle midzone coordinately directs formation of the cytokinetic furrow. Here, we explore the contribution of the asters by analyzing the consequences of altering interaster distance during the first cytokinesis of the Caenorhabditis elegans embryo. Delaying aster separation, by using TPXL-1 depletion to shorten the metaphase spindle, leads to a corresponding delay in furrow formation, but results in a single furrow that ingresses at a normal rate. Preventing aster separation, by simultaneously inhibiting TPXL-1 and Gα signaling-based cortical forces pulling on the asters, delays furrow formation and leads to the formation of multiple furrows that ingress toward the midzone. Disrupting midzone-based signaling, by depleting conserved midzone complexes, results in a converse phenotype: neither the timing nor the number of furrows is affected, but the rate of furrow ingression is decreased threefold. Simultaneously delaying aster separation and disrupting midzone-based signaling leads to complete failure of furrow formation. Based on these results, we propose that signaling by the separated asters executes two critical functions: 1) it couples furrow formation to anaphase onset by concentrating contractile ring proteins on the equatorial cortex in a midzone-independent manner and 2) it subsequently refines spindle midzone-based signaling to restrict furrowing to a single site.     

A Highlights from MBoC Selection: CGEF-1 and CHIN-1 Regulate CDC-42 Activity during Asymmetric Division in the Caenorhabditis elegans Embryo

The anterior–posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.     

A Highlights from MBoC Selection: Slk19 clusters kinetochores and facilitates chromosome bipolar attachment

In all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome segregation has remained elusive. Here we show that Slk19 is required for kinetochore clustering when yeast cells are treated with the microtubule-depolymerizing agent nocodazole. We further find that slk19Δ mutant cells exhibit delayed kinetochore capture and chromosome bipolar attachment after the disruption of the kinetochore–microtubule interaction by nocodazole, which is likely attributed to defective kinetochore clustering. In addition, we show that Slk19 interacts with itself, suggesting that the dimerization of Slk19 may mediate the interaction between kinetochores for clustering. Therefore Slk19 likely acts as kinetochore glue that clusters kinetochores to facilitate efficient and faithful chromosome segregation.     

A Highlights from MBoC Selection: Fast Microtubule Dynamics in Meiotic Spindles Measured by Single Molecule Imaging: Evidence That the Spindle Environment Does Not Stabilize Microtubules

Metaphase spindles are steady-state ensembles of microtubules that turn over rapidly and slide poleward in some systems. Since the discovery of dynamic instability in the mid-1980s, models for spindle morphogenesis have proposed that microtubules are stabilized by the spindle environment. We used single molecule imaging to measure tubulin turnover in spindles, and nonspindle assemblies, in Xenopus laevis egg extracts. We observed many events where tubulin molecules spend only a few seconds in polymer and thus are difficult to reconcile with standard models of polymerization dynamics. Our data can be quantitatively explained by a simple, phenomenological model—with only one adjustable parameter—in which the growing and shrinking of microtubule ends is approximated as a biased random walk. Microtubule turnover kinetics did not vary with position in the spindle and were the same in spindles and nonspindle ensembles nucleated by Tetrahymena pellicles. These results argue that the high density of microtubules in spindles compared with bulk cytoplasm is caused by local enhancement of nucleation and not by local stabilization. It follows that the key to understanding spindle morphogenesis will be to elucidate how nucleation is spatially controlled.     

A Highlights from MBoC Selection: Locus-specific gene repositioning in prostate cancer

Genes occupy preferred spatial positions within interphase cell nuclei. However, positioning patterns are not an innate feature of a locus, and genes can alter their localization in response to physiological and pathological changes. Here we screen the radial positioning patterns of 40 genes in normal, hyperplasic, and malignant human prostate tissues. We find that the overall spatial organization of the genome in prostate tissue is largely conserved among individuals. We identify three genes whose nuclear positions are robustly altered in neoplastic prostate tissues. FLI1 and MMP9 position differently in prostate cancer than in normal tissue and prostate hyperplasia, whereas MMP2 is repositioned in both prostate cancer and hyperplasia. Our data point to locus-specific reorganization of the genome during prostate disease.     

A Highlights from MBoC Selection: Systematic identification of pathological lamin A interactors

Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype–phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A–binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome–wide ORFeome library, we identified and validated 337 lamin A–binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)–like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein–protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.     

A Highlights from MBoC Selection: Load adaptation by endocytic actin networks

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly–mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity, we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.     

A Highlights from MBoC Selection: mTOR regulates phagosome and entotic vacuole fission

Macroendocytic vacuoles formed by phagocytosis, or the live-cell engulfment program entosis, undergo sequential steps of maturation, leading to the fusion of lysosomes that digest internalized cargo. After cargo digestion, nutrients must be exported to the cytosol, and vacuole membranes must be processed by mechanisms that remain poorly defined. Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks. Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells. Degrading engulfed cells supply engulfing cells with amino acids that are used in translation, and rescue cell survival and mTORC1 activity in starved macrophages and tumor cells. These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.     

A Highlights from MBoC Selection: Nebulin binding impedes mutant desmin filament assembly

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160–164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.     

A Highlights from MBoC Selection: Myosin Vs organize actin cables in fission yeast

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.     

A Highlights from MBoC Selection: Regulation of microtubule-based transport by MAP4

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.     

A Highlights from MBoC Selection: Nucleotide- and Protein-Dependent Functions of Actg1

Cytoplasmic β- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences Actb and Actg1, respectively, are more divergent but still 89% similar. Actb^–/– mice are embryonic lethal and Actb^–/– cells fail to proliferate, but editing the Actb gene to express γ-actin (Actb^c–g) resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for Actb. To determine if Actg1 has a protein-independent function, we crossed Actb^c–g and Actg1^–/– mice to generate the bG/0 line, where the only cytoplasmic actin expressed is γ-actin from Actb^c–g. The bG/0 mice were viable but showed a survival defect despite expressing γ-actin protein at levels no different from bG/gG with normal survival. A unique myopathy phenotype was also observed in bG/0 mice. We conclude that impaired survival and myopathy in bG/0 mice are due to loss of Actg1 nucleotide-dependent function(s). On the other hand, the bG/0 genotype rescued functions impaired by Actg1^–/–, including cell proliferation and auditory function, suggesting a role for γ-actin protein in both fibroblasts and hearing. Together, these results identify nucleotide-dependent functions for Actg1 while implicating γ-actin protein in more cell-/tissue-specific functions.     

A Highlights from MBoC Selection: Persistent growth of microtubules at low density

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability—transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density–dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.