Xiao Wei Chen

A novel Crumbs3 isoform regulates cell division and ciliogenesis via importin β interactions

The Crumbs family of apical transmembrane proteins regulates apicobasal polarity via protein interactions with a conserved C-terminal sequence, ERLI. However, one of the mammalian Crumbs proteins, Crumbs3 (CRB3) has an alternate splice form with a novel C-terminal sequence ending in CLPI (CRB3-CLPI). We report that CRB3-CLPI localizes to the cilia membrane and a membrane compartment at the mitotic spindle poles. Knockdown of CRB3-CLPI leads to both a loss of cilia and a multinuclear phenotype associated with centrosomal and spindle abnormalities. Using protein purification, we find that CRB3-CLPI interacts with importin β-1 in a Ran-regulated fashion. Importin β-1 colocalizes with CRB3-CLPI during mitosis, and a dominant-negative form of importin β-1 closely phenocopies CRB3-CLPI knockdown. Knockdown of importin β-1 blocks targeting of CRB3-CLPI to the spindle poles. Our data suggest an expanded role for Crumbs proteins in polarized membrane targeting and cell division via unique interactions with importin proteins.

RalA-exocyst-dependent recycling endosome trafficking is required for the completion of cytokinesis.

In eukaryotic cells, recycling endosome-mediated trafficking contributes to the completion of cytokinesis, in a manner under the control of the centrosome. We report that the exocyst complex and its interacting GTPase RalA play a critical role in this polarized trafficking process. RalA resides in the recycling endosome and relocates from the pericentrosomal region to key cytokinetic structures including the cleavage furrow, and later, the abscission site. This event is coupled to the dynamic redistribution of the exocyst proteins. These associate with the centrosome in interphase and concentrate on the central spindle/midbody during cytokinesis. Disruption of RalA-exocyst function leads to cytokinesis failure in late stages, particularly abscission, resembling the cytokinesis defects induced by loss of centrosome function. These data suggest that RalA and the exocyst may regulate vesicle delivery to the centrosome-related abscission site during the terminal stage of cytokinesis, implicating RalA as a critical regulator of cell cycle progression.

A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

It is shown that RalA is regulated by a Ral GAP complex (RGC 1/2) in insulin action and links PI 3-kinase signaling to RalA activation. Akt phosphorylates the complex and inhibits its function, resulting in increased RalA activity and glucose uptake.

Structural Basis of Ist1 Function and Ist1–Did2 Interaction in the Multivesicular Body Pathway and Cytokinesis

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.

SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI: http://dx.doi.org/10.7554/eLife.00444.001

SEC23B is required for the maintenance of murine professional secretory tissues

In eukaryotic cells, newly synthesized secretory proteins require COPII (coat protein complex II) to exit the endoplasmic reticulum (ER). COPII contains five core components: SAR1, SEC23, SEC24, SEC13, and SEC31. SEC23 is a GTPase-activating protein that activates the SAR1 GTPase and also plays a role in cargo recognition. Missense mutations in the human COPII paralogues SEC23A and SEC23B result in craniolenticulosutural dysplasia and congenital dyserythropoietic anemia type II, respectively. We now report that mice completely deficient for SEC23B are born with no apparent anemia phenotype, but die shortly after birth, with degeneration of professional secretory tissues. In SEC23B-deficient embryonic pancreas, defects occur in exocrine and endocrine tissues shortly after differentiation. Pancreatic acini are completely devoid of zymogen granules, and the ER is severely distended. Similar ultrastructural alterations are also observed in salivary glands, but not in liver. Accumulation of proteins in the ER lumen activates the proapoptotic pathway of the unfolded protein response, suggesting a central role for apoptosis in the degeneration of these tissues in SEC23B-deficient embryos. Although maintenance of the secretory pathway should be required by all cells, our findings reveal a surprising tissue-specific dependence on SEC23B for the ER exit of highly abundant cargo, with high levels of SEC23B expression observed in professional secretory tissues. The disparate phenotypes in mouse and human could result from residual SEC23B function associated with the hypomorphic mutations observed in humans, or alternatively, might be explained by a species-specific shift in function between the closely related SEC23 paralogues.

CAP interacts with cytoskeletal proteins and regulates adhesion‐mediated ERK activation and motility

CAP/Ponsin belongs to the SoHo family of adaptor molecules that includes ArgBP2 and Vinexin. These proteins possess an N‐terminal sorbin homology (SoHo) domain and three C‐terminal SH3 domains that bind to diverse signaling molecules involved in a variety of cellular processes. Here, we show that CAP binds to the cytoskeletal proteins paxillin and vinculin. CAP localizes to cell–extracellular matrix (ECM) adhesion sites, and this process requires binding to vinculin. Overexpression of CAP induces the aggregation of paxillin, vinculin and actin at cell–ECM adhesion sites. Moreover, CAP inhibits adhesion‐dependent processes such as cell spreading and focal adhesion turnover, whereas a CAP mutant that is unable to localize to cell–ECM adhesion sites is incapable of exerting these effects. Finally, depletion of CAP by siRNA‐mediated knockdown leads to enhanced cell spreading, migration and the activation of the PAK/MEK/ERK pathway in REF52 cells. Taken together, these results indicate that CAP is a cytoskeletal adaptor protein involved in modulating adhesion‐mediated signaling events that lead to cell migration.

SRA Regulates Adipogenesis by Modulating p38/JNK Phosphorylation and Stimulating Insulin Receptor Gene Expression and Downstream Signaling

The Steroid Receptor RNA Activator (SRA) enhances adipogenesis and increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. To assess the mechanism, we differentiated ST2 mesenchymal precursor cells that did or did not overexpress SRA into adipocytes using combinations of methylisobutylxanthine, dexamethasone and insulin. These studies showed that SRA overexpression promotes full adipogenesis in part by stimulation of insulin/insulin-like growth factor-1 (IGF-1) signaling. SRA overexpression inhibited phosphorylation of p38 mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) in the early differentiation of ST2 cells. Conversely, knockdown of endogenous SRA in 3T3-L1 cells increased phosphorylation of JNK. Knockdown of SRA in mature 3T3-L1 adipocytes reduced insulin receptor (IR) mRNA and protein levels, which led to decreased autophosphorylation of IRβ and decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt. This likely reflects a stimulatory role of SRA on IR transcription, as transfection studies showed that SRA increased expression of an IR promoter-luciferase reporter construct.

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes

Rab10 can increase insulin-stimulated activation of RalA in 3T3-L1 adipocytes. Rlf is a new effector of Rab10 and is required for maximal glucose uptake in adipocytes. Rab10 recruits Rlf to activate RalA.

Circadian Metabolic Regulation through Crosstalk between Casein Kinase 1δ and Transcriptional Coactivator PGC-1α

Circadian clock coordinates behavior and physiology in mammals in response to light and feeding cycles. Disruption of normal clock function is associated with increased risk for cardiovascular and metabolic diseases, underscoring the emerging concept that temporal regulation of tissue metabolism is a fundamental aspect of energy homeostasis. We have previously demonstrated that transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), coordinates circadian metabolic rhythms through simultaneous regulation of metabolic and clock gene expression. In this study, we found that PGC-1α physically interacts with, and is phosphorylated by, casein kinase 1δ (CK1δ), a core component of the circadian pacemaker. CK1δ represses the transcriptional function of PGC-1α in cultured hepatocytes, resulting in decreased gluconeogenic gene expression and glucose secretion. At the molecular level, CK1δ phosphorylation of PGC-1α within its arginine/serine-rich domain enhances its degradation through the proteasome system. Together, these results elucidate a novel mechanism through which circadian pacemaker transduces timing signals to the metabolic regulatory network that controls hepatic energy metabolism.