scholarly journals FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility in mice

Development ◽  
2021 ◽  
Vol 148 (21) ◽  
Author(s):  
Akane Morohoshi ◽  
Haruhiko Miyata ◽  
Yuki Oyama ◽  
Seiya Oura ◽  
Taichi Noda ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Male Fertility ◽  
Morphological Changes ◽  
Vesicle Trafficking ◽  
Sequence Similarity ◽  
Active Form ◽  
Small Gtpase ◽  
Mutant Mice ◽  
Mass Spectrometry Analysis ◽  
Acrosome Formation

ABSTRACT The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

scholarly journals Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 172
Author(s):  
Steen Vang Petersen ◽  
Nanna Bach Poulsen ◽  
Cecilie Linneberg Matthiesen ◽  
Frederik Vilhardt
Keyword(s):  
Hydrogen Peroxide ◽  
Membrane Trafficking ◽  
Spatial Organization ◽  
Redox Signaling ◽  
Small Gtpase ◽  
Paracrine Signaling ◽  
Tissue Macrophage ◽  
Storage Vesicles ◽  
Superoxide Dismutase 3 ◽  
Macrophage Populations

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

scholarly journals Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice

2019 ◽  
Vol 116 (37) ◽  
pp. 18498-18506 ◽  
Author(s):  
Yoshitaka Fujihara ◽  
Taichi Noda ◽  
Kiyonori Kobayashi ◽  
Asami Oji ◽  
Sumire Kobayashi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Male Fertility ◽  
Reproductive Tract ◽  
Gene Families ◽  
Gene Clusters ◽  
Mutant Mice ◽  
Male Reproductive Tract ◽  
Sperm Migration ◽  
Specific Proteins ◽  
Multiple Male

CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. AlthoughGdpd1andGdpd4mutant mice were fertile, disruptions ofCstandPategene clusters andLypd4resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

scholarly journals The early endosomal protein Rab21 is critical for enterocyte functions and intestinal homeostasis

2020 ◽  
Author(s):  
Sonya Nassari ◽  
Dominique Lévesque ◽  
François-Michel Boisvert ◽  
Steve Jean
Keyword(s):  
Membrane Trafficking ◽  
Genetic Diseases ◽  
Growth Factor Receptor ◽  
Small Gtpase ◽  
Intestinal Morphology ◽  
Endosomal Trafficking ◽  
Intestinal Homeostasis ◽  
Compensatory Proliferation ◽  
Epidermal Growth

ABSTRACTMembrane trafficking is defined as the vesicular transport of molecules into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to genetic diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo model system, we investigated enterocyte-specific functions for the early endosomal trafficking machinery in gut homeostasis. We focused on the small GTPase Rab21, which regulates specific steps in early endosomal trafficking. Rab21-depleted guts showed severe abnormalities in intestinal morphology, with deregulated homeostasis associated with a gain in mitotic cells and increased cell death. Increases in both apoptosis and yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using a RNA interference screen, we identified specific regulators of autophagy and membrane trafficking that phenocopied Rab21 loss. We further showed that Rab21-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling, and identified improperly trafficked cargoes in Rab21-depleted enterocytes. Our data shed light on an important role for early endosomal trafficking, and particularly Rab21, in enterocyte-mediated intestinal homeostasis.

scholarly journals A Phytophthora capsici RXLR effector manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein trafficking pathway

2021 ◽  
Author(s):  
Tianli Li ◽  
Gan Ai ◽  
Xiaowei Fu ◽  
Jin Liu ◽  
Hai Zhu ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Plant Immunity ◽  
Phytophthora Capsici ◽  
Functional Characterization ◽  
Ectopic Expression ◽  
Defense Responses ◽  
Infection Process ◽  
Small Gtpase ◽  
Rab Proteins ◽  
Rxlr Effector

The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors to enter plant cells and suppress host defense responses. Only few of them are conserved across different strains and species. Such ‘core effectors’ may target hub immunity pathways that are essential during Phytophthora pathogens interacting with their hosts. However, the underlying mechanisms of core RXLRs-mediated host immunity manipulation are largely unknown. Here, we report the functional characterization of a P. capsici RXLR effector, RXLR242. RXLR242 expression is highly induced during the infection process. Its ectopic expression in Nicotiana benthamiana promotes Phytophthora infection. RXLR242 physically interacts with a group of RAB proteins, which belong to the small GTPase family and function in specifying transport pathways in the intracellular membrane trafficking system. RXLR242 impedes the secretion of PATHOGENESIS-RELATED 1 (PR1) protein to the apoplast by interfering the formation of RABE1-7-labeled vesicles. Further analysis indicated that such phenomenon is resulted from competitive binding of RXLR242 to RABE1-7. RXLR242 also interferes trafficking of the membrane-located receptor FLAGELLIN-SENSING 2 (FLS2) through competitively interacting with RABA4-3. Taken together, our work demonstrates that RXLR242 manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein transporting pathway in plant hosts.

scholarly journals Distinct Roles for the Yeast Phosphatidylinositol 4-Kinases, Stt4p and Pik1p, in Secretion, Cell Growth, and Organelle Membrane Dynamics

2000 ◽  
Vol 11 (8) ◽  
pp. 2673-2689 ◽  
Author(s):  
Anjon Audhya ◽  
Michelangelo Foti ◽  
Scott D. Emr
Keyword(s):  
Cell Growth ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoskeleton Organization ◽  
Yeast Viability

The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases,STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 orPIK1 is lethal. Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth. By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization. In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis. Strikingly,pik1tscells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4tscells exhibit no detectable secretory defects. Both single mutants reduce PtdIns(4)P by ∼50%; however,stt4ts/pik1tsdouble mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P2. The aberrant Golgi morphology found in pik1tsmutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell. Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels. In contrast, diminished levels of PtdIns(4)P observed in stt4tscells at restrictive temperature result in a dramatic change in vacuole size compared with pik1tscells and persistent actin delocalization. Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P2that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.

scholarly journals Calcium-sensing receptor deletion in the mouse esophagus alters barrier function

2020 ◽  
Vol 318 (1) ◽  
pp. G144-G161 ◽  
Author(s):  
Nazih L. Nakhoul ◽  
Chia-Ling Tu ◽  
Karen L. Brown ◽  
M. Toriqul Islam ◽  
Anna G. Hodges ◽  
...  
Keyword(s):  
Barrier Function ◽  
Morphological Changes ◽  
Ussing Chamber ◽  
Small Gtpase ◽  
Pcr Analysis ◽  
Basal Cells ◽  
Calcium Sensing Receptor ◽  
Epithelial Surface ◽  
Calcium Sensing ◽  
Junction Protein

Calcium-sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca2+ concentrations. CaSR has been reported to play a role in glandular and fluid secretion in the gastrointestinal tract and to regulate differentiation and proliferation of skin keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating keratin 5 CreER;CaSRFlox+/+compound mutants, in which loxP sites flank exon 7 of CaSR gene. Recombination was initiated with multiple tamoxifen injections, and we demonstrated exon 7 deletion by PCR analysis of genomic DNA. Quantitative real-time PCR and Western blot analyses showed a significant reduction in CaSR mRNA and protein expression in the knockout mice ( EsoCaSR−/−) as compared with control mice. Microscopic examination of EsoCaSR−/− esophageal tissues showed morphological changes including elongation of the rete pegs, abnormal keratinization and stratification, and bacterial buildup on the luminal epithelial surface. Western analysis revealed a significant reduction in levels of adherens junction proteins E-cadherin and β catenin and tight junction protein claudin-1, 4, and 5. Levels of small GTPase proteins Rac/Cdc42, involved in actin remodeling, were also reduced. Ussing chamber experiments showed a significantly lower transepithelial resistance in knockout (KO) tissues. In addition, luminal-to-serosal-fluorescein dextran (4 kDa) flux was higher in KO tissues. Our data indicate that CaSR plays a role in regulating keratinization and cell-cell junctional complexes and is therefore important for the maintenance of the barrier function of the esophagus. NEW & NOTEWORTHY The esophageal stratified squamous epithelium maintains its integrity by continuous proliferation and differentiation of the basal cells. Here, we demonstrate that deletion of the calcium-sensing receptor, a G protein-coupled receptor, from the basal cells disrupts the structure and barrier properties of the epithelium.

A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5052-5062 ◽  
Author(s):  
Gaël Ménasché ◽  
Mickaël M. Ménager ◽  
Juliette M. Lefebvre ◽  
Einat Deutsch ◽  
Rafika Athman ◽  
...  
Keyword(s):  
Target Cell ◽  
Affinity Purification ◽  
Cytotoxic T Cells ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Granule Secretion ◽  
Immunologic Synapse ◽  
Cytotoxic Granule

Abstract Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules—indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B

2001 ◽  
Vol 356 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Mireille CORMONT ◽  
Nadine GAUTIER ◽  
Karine ILC ◽  
Yannick Le MARCHAND-BRUSTEL
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Phosphatidylinositol 3 Kinase ◽  
Isolated Adipocytes ◽  
Phosphatidylinositol 3

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

The Small GTPase Superfamily in Plants: A Conserved Regulatory Module with Novel Functions

2020 ◽  
Vol 71 (1) ◽  
pp. 247-272
Author(s):  
Erik Nielsen
Keyword(s):  
Membrane Trafficking ◽  
Binding Proteins ◽  
Model Organism ◽  
Nucleocytoplasmic Transport ◽  
Small Gtpase ◽  
Gtp Binding Proteins ◽  
Cellular Processes ◽  
Gtp Binding ◽  
Regulatory Module ◽  
Transport Vesicle

Small GTP-binding proteins represent a highly conserved signaling module in eukaryotes that regulates diverse cellular processes such as signal transduction, cytoskeletal organization and cell polarity, cell proliferation and differentiation, intracellular membrane trafficking and transport vesicle formation, and nucleocytoplasmic transport. These proteins function as molecular switches that cycle between active and inactive states, and this cycle is linked to GTP binding and hydrolysis. In this review, the roles of the plant complement of small GTP-binding proteins in these cellular processes are described, as well as accessory proteins that control their activity, and current understanding of the functions of individual members of these families in plants—with a focus on the model organism Arabidopsis—is presented. Some potential novel roles of these GTPases in plants, relative to their established roles in yeast and/or animal systems, are also discussed.

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