A Highlights from MBoC Selection: Rab21 in enterocytes participates in intestinal epithelium maintenance

Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery. We focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, we identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. We further showed that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, we validated changes in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. Our data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance.     

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: Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

C2 domains are widespread motifs that often serve as Ca²⁺-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca²⁺ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca²⁺, and shifted the Ca²⁺ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.     

A Highlights from MBoC Selection: Golgi Partitioning Controls Mitotic Entry through Aurora-A Kinase

At the onset of mitosis, the Golgi complex undergoes a multistep fragmentation process that is required for its correct partitioning into the daughter cells. Inhibition of this Golgi fragmentation results in cell cycle arrest at the G2 stage, suggesting that correct inheritance of the Golgi complex is monitored by a “Golgi mitotic checkpoint.” However, the molecular basis of this G2 block is not known. Here, we show that the G2-specific Golgi fragmentation stage is concomitant with centrosome recruitment and activation of the mitotic kinase Aurora-A, an essential regulator for entry into mitosis. We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression. Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.     

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: 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: Structural basis for recognition of the Sec4 Rab GTPase by its effector,the Lgl/tomosyn homologue,Sro7

Members of the tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in tomosyn vertebrate homologues.     

A Highlights from MBoC Selection: Rab11-family interacting proteins define spatially and temporally distinct regions within the dynamic Rab11a-dependent recycling system

The Rab11-family interacting proteins (Rab11-FIPs) facilitate Rab11-dependent vesicle recycling. We hypothesized that Rab11-FIPs define discrete subdomains and carry out temporally distinct roles within the recycling system. We used live-cell deconvolution microscopy of HeLa cells expressing chimeric fluorescent Rab11-FIPs to examine Rab11-FIP localization, transferrin passage through Rab11-FIP–containing compartments, and overlap among Rab11-FIPs within the recycling system. FIP1A, FIP2, and FIP5 occupy widely distributed mobile tubules and vesicles, whereas FIP1B, FIP1C, and FIP3 localize to perinuclear tubules. Internalized transferrin entered Rab11-FIP–containing compartments within 5 min, reaching maximum colocalization with FIP1B and FIP2 early in the time course, whereas localization with FIP1A, FIP1C, FIP3, and FIP5 was delayed until 10 min or later. Whereas direct interactions with FIP1A were only observed for FIP1B and FIP1C, FIP1A also associated with membranes containing FIP3. Live-cell dual-expression studies of Rab11-FIPs revealed the tubular dynamics of Rab11-FIP–containing compartments and demonstrated a series of selective associations among Rab11-FIPs in real time. These findings suggest that Rab11-FIP1 proteins participate in spatially and temporally distinct steps of the recycling process along a complex and dynamic tubular network in which Rab11-FIPs occupy discrete domains.     

A Highlights from MBoC Selection: Insulin-promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.     

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: 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: 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: 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: Multiple protein kinases influence the redistribution of fission yeast Clp1/Cdc14 phosphatase upon genotoxic stress

The Cdc14 phosphatase family antagonizes Cdk1 phosphorylation and is important for mitotic exit. To access their substrates, Cdc14 phosphatases are released from nucleolar sequestration during mitosis. Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 orthologue, and Cdc14B, a mammalian orthologue, also exit the nucleolus during interphase upon DNA replication stress or damage, respectively, implicating Cdc14 phosphatases in the response to genotoxic insults. However, a mechanistic understanding of Cdc14 phosphatase nucleolar release under these conditions is incomplete. We show here that relocalization of Clp1 during genotoxic stress is governed by complex phosphoregulation. Specifically, the Rad3 checkpoint effector kinases Cds1 and/or Chk1, the cell wall integrity mitogen-activated protein kinase Pmk1, and the cell cycle kinase Cdk1 directly phosphorylate Clp1 to promote genotoxic stress–induced nucleoplasmic accumulation. However, Cds1 and/or Chk1 phosphorylate RxxS sites preferentially upon hydroxyurea treatment, whereas Pmk1 and Cdk1 preferentially phosphorylate Clp1 TP sites upon H₂O₂ treatment. Abolishing both Clp1 RxxS and TP phosphosites eliminates any genotoxic stress–induced redistribution. Reciprocally, preventing dephosphorylation of Clp1 TP sites shifts the distribution of the enzyme to the nucleoplasm constitutively. This work advances our understanding of pathways influencing Clp1 localization and may provide insight into mechanisms controlling Cdc14B phosphatases in higher eukaryotes.     

A Highlights from MBoC Selection: A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells

Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a–Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.     

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.