Sar1 GTPase Activity Is Regulated by Membrane Curvature.

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5'-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.This work was supported by grants from the NIH (GM110567 and GM088151 to AA). IM, RMH and JME were supported by a grant from the Biotechnology and Biological Sciences Research Council (BB/J018236/1). ERC is an Investigator of the Howard Hughes Medical Institute. We thank Elizabeth Miller for providing purified yeast COPII components, Subhanjan Mondal and Said Goueli at Promega Corporation for providing us access to the GTPase-Glo system ahead of release, and members of the Audhya lab for critically reading this manuscript.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.67228

A role for Rab5 in structuring the endoplasmic reticulum

The endoplasmic reticulum (ER) is a contiguous network of interconnected membrane sheets and tubules. The ER is differentiated into distinct domains, including the peripheral ER and nuclear envelope. Inhibition of two ER proteins, Rtn4a and DP1/NogoA, was previously shown to inhibit the formation of ER tubules in vitro. We show that the formation of ER tubules in vitro also requires a Rab family GTPase. Characterization of the 29 Caenorhabditis elegans Rab GTPases reveals that depletion of RAB-5 phenocopies the defects in peripheral ER structure that result from depletion of RET-1 and YOP-1, the C. elegans homologues of Rtn4a and DP1/NogoA. Perturbation of endocytosis by other means did not affect ER structure; the role of RAB-5 in ER morphology is thus independent of its well-studied requirement for endocytosis. RAB-5 and YOP-1/RET-1 also control the kinetics of nuclear envelope disassembly, which suggests an important role for the morphology of the peripheral ER in this process

PENGARUH ASET LAIN-LAIN DAN POLITIK DINASTI TERHADAP AUDIT DELAY PADA PEMERINTAH DAERAH KABUPATEN/KOTA TAHUN 2021

Penelitian ini bertujuan untuk mengetahui pengaruh aset lain-lain dan politik dinasti terhadap audit delay. Populasi penelitian ini adalah pemerintah daerah kabupaten/kota di Indonesia tahun 2021 dengan metode pengambilan sampel menggunakan teknik purposive sampling sehingga didapatkan sampel sebanyak 166 pemerintah daerah. Teknik analisis yang digunakan dalam melakukan uji hipotesis yaitu analisis regresi linear berganda. Hasil pengujian hipotesis menunjukkan bahwa (1) aset lain-lain tidak berpengaruh terhadap audit delay; dan (2) politik dinasti berpengaruh positif terhadap audit delay. This study aims to determine the effect of other assets and dynastic politics on audit delay. The population for this research is district/city local governments in Indonesia in 2021. The sampling method uses a purposive sampling technique so that a sample of 166 local governments is obtained. The analysis technique used in testing the hypothesis is multiple linear regression analysis. The results of hypothesis testing show that (1) other assets have no effect on audit delay; and (2) dynastic politics has a positive effect on audit delay

Micro-proteomics with iterative data analysis:proteome analysis in <i>C.elegans</i> at the single worm level

Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and approximate to 1500 germ cells, measuring the worm proteome at a single organism level to a depth of approximate to 3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (&gt;6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (), an open access, searchable database resource

Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3

The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling

Katanin controls mitotic and meiotic spindle length

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that γ-tubulin–dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly

A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans

Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1–depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis

Caregiver Global Impression Observations from EMBARK: A Phase 3 Study Evaluating Delandistrogene Moxeparvovec in Ambulatory Patients with Duchenne Muscular Dystrophy.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is a rare, progressive, debilitating neuromuscular disease. The early childhood onset and debilitating nature of the disease necessitate decades of caretaking for most patients. Caregivers have a critical role in evaluating patients physical functioning and/or response to treatment. Using DMD-specific caregiver-reported scales, the impact of delandistrogene moxeparvovec gene therapy on caregivers perceived change in patient disease status or severity was evaluated using the Caregiver Global Impression of Change and Severity (CaGI-C and CaGI-S, respectively). METHODS: In the Phase 3 randomized, double-blind, placebo-controlled trial (EMBARK; NCT05096221), the CaGI-C at week 52 and change from baseline to week 52 in CaGI-S were evaluated in a post hoc analysis. The CaGI-C assesses caregivers impressions of change in DMD symptoms, physical ability, ability to perform daily activities, and overall health. The CaGI-S evaluates current severity of DMD symptoms, physical ability, ability to perform activities of daily living, and overall health. Data were evaluated using multi-domain responder index (MDRI) and ordinal regression analyses. RESULTS: MDRI analyses across all four CaGI-C items yielded a treatment difference of 1.7 (95% confidence interval [CI]: 0.90-2.5) favoring delandistrogene moxeparvovec; a treatment difference of 1.1 (95% CI 0.30-1.9) was observed for the CaGI-S favoring delandistrogene moxeparvovec. After adjusting for age, ordinal regression analysis showed a nominally significant increase in the odds of achieving a better rating for delandistrogene moxeparvovec-treated patients on all four CaGI-C items (≥ 3.8-fold increase). After adjusting for baseline severity and age, ordinal regression analysis showed a nominally significant increase in the odds of improvement on all four CaGI-S items (≥ 2.2-fold increase). CONCLUSION: These exploratory findings captured by caregiver-reported outcomes add to the totality of evidence that supports the clinical benefits of delandistrogene moxeparvovec for patients with DMD. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier, NCT05096221

UMA and MABP domains throw light on receptor endocytosis and selection of endosomal cargoes

Interactions of the ESCRT complexes are critical for endosomal trafficking. We identify two domains with potential significance for this process. The MABP domain present in metazoan ESCRT-I/MVB12 subunits, Crag, a regulator of protein sorting, and bacterial pore-forming proteins might mediate novel membrane interactions in trafficking. The UBAP1-MVB12-associated UMA domain found in MVB12 and UBAP1 defines a novel adaptor that might recruit diverse targets to ESCRT-I