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: A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction

Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR–domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation–promoting factor N-WASP to tight junctions. CRISPR-Cas9–mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1–knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin.     

A Highlights from MBoC Selection: Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles

Sorting of plasma membrane proteins into exocytic vesicles at the yeast trans-Golgi network (TGN) is believed to be mediated by their coalescence with specific lipids, but how these membrane-remodeling events are regulated is poorly understood. Here we show that the ATP-dependent phospholipid flippase Drs2 is required for efficient segregation of cargo into exocytic vesicles. The plasma membrane proteins Pma1 and Can1 are missorted from the TGN to the vacuole in drs2∆ cells. We also used a combination of flippase mutants that either gain or lose the ability to flip phosphatidylserine (PS) to determine that PS flip by Drs2 is its critical function in this sorting event. The primary role of PS flip at the TGN appears to be to control the oxysterol-binding protein homologue Kes1/Osh4 and regulate ergosterol subcellular distribution. Deletion of KES1 suppresses plasma membrane–missorting defects and the accumulation of intracellular ergosterol in drs2 mutants. We propose that PS flip is part of a homeostatic mechanism that controls sterol loading and lateral segregation of protein and lipid domains at the TGN.     

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: Nuclear LSm8 affects number of cytoplasmic processing bodies via controlling cellular distribution of Like-Sm proteins

Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2–7 proteins. We propose a model in which redistribution of LSm2–7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies.     

A Highlights from MBoC Selection: LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon development

The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.     

A Highlights from MBoC Selection: MAP65/Ase1 promote microtubule flexibility

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1–8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.     

A Highlights from MBoC Selection: Discovery of a vezatin-like protein for dynein-mediated early endosome transport

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo.     

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: Coordination of Grp1 recruitment mechanisms by its phosphorylation

The action of guanine nucleotide exchange factors (GEFs) on the ADP-ribosylation factor (ARF) family of small GTPases initiates intracellular transport pathways. This role requires ARF GEFs to be recruited from the cytosol to intracellular membrane compartments. An ARF GEF known as General receptor for 3-phosphoinositides 1 (Grp1) is recruited to the plasma membrane through its pleckstrin homology (PH) domain that recognizes phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we find that the phosphorylation of Grp1 induces its PH domain to recognize instead phosphatidylinositol 4-phosphate (PI4P). This phosphorylation also releases an autoinhibitory mechanism that results in the coil–coil (CC) domain of Grp1 engaging two peripheral membrane proteins of the recycling endosome. Because the combination of these actions results in Grp1 being recruited preferentially to the recycling endosome rather than to the plasma membrane, our findings reveal the complexity of recruitment mechanisms that need to be coordinated in localizing an ARF GEF to an intracellular compartment to initiate a transport pathway. Our elucidation is also remarkable for having revealed that phosphoinositide recognition by a PH domain can be switched through its phosphorylation.     

A Highlights from MBoC Selection: The Late Endosome is Essential for mTORC1 Signaling

The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid–stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2−/− MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid– and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.     

A Highlights from MBoC Selection: Drosha protein levels are translationally regulated during Xenopus oocyte maturation

MicroRNAs (miRNAs) are ∼21-nucleotide-long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes, Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous primary miRNAs (pri-miRNAs) is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly(A) length addition to the Drosha mRNA, which boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine-to-inosine deamination–type RNA editing. Using activated Xenopus eggs for microinjection experiments, we demonstrate that RNA editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA editing.     

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: p52Shc regulates the sustainability of ERK activation in a RAF-independent manner

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.