scholarly journals Cdc42 GTPase activating proteins Rga4 and Rga6 coordinate septum synthesis and membrane trafficking at the division plane during cytokinesis

2021 ◽  
Author(s):  
Bethany F Campbell ◽  
Brian S Hercyk ◽  
Ashlei R Williams ◽  
Emalyn S San Miguel ◽  
Haylee G Young ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Membrane Vesicles ◽  
Membrane Structures ◽  
Division Plane ◽  
Septum Formation ◽  
Division Site ◽  
Gtpase Activating Proteins ◽  
Primary Septum ◽  
Separate Electron ◽  
Specific Membrane

Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. In rga4Δrga6Δ mutants Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates only specific membrane trafficking events. Our data indicate that while active Cdc42 promotes primary septum synthesis, as cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity. This couples specific membrane trafficking events with septum formation to allow proper septum morphology.

scholarly journals Cdc42 promotes Bgs1 recruitment for septum synthesis and glucanase localization for cell separation during cytokinesis in fission yeast

2019 ◽  
Author(s):  
Udo N. Onwubiko ◽  
Julie Robinson ◽  
Rose Albu Mustaf ◽  
Maitreyi E. Das
Keyword(s):  
Fission Yeast ◽  
Membrane Trafficking ◽  
Cell Separation ◽  
Nucleotide Exchange ◽  
Timely Initiation ◽  
Division Site ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Primary Septum ◽  
Ring Constriction

AbstractCytokinesis in fission yeast involves actomyosin ring constriction concurrent to septum synthesis followed by septum digestion resulting in cell separation. A recent report indicates that endocytosis is required for septum synthesis and cell separation. The conserved GTPase Cdc42 is required for membrane trafficking and promotes endocytosis. Cdc42 is activated by Guanine nucleotide exchange factors (GEFs). Cdc42 GEFs have been shown to promote timely initiation of septum synthesis and proper septum morphology. Here we show that Cdc42 promotes the recruitment of the major primary septum synthesizing enzyme Bgs1 and consequent ring constriction. Cdc42 is also required for proper localization of the septum digesting glucanases at the division site. Thus, Cdc42 is required to promote multiple steps during cytokinesis.

scholarly journals Functional dissection of Rab GTPases involved in primary cilium formation

2007 ◽  
Vol 178 (3) ◽  
pp. 363-369 ◽  
Author(s):  
Shin-ichiro Yoshimura ◽  
Johannes Egerer ◽  
Evelyn Fuchs ◽  
Alexander K. Haas ◽  
Francis A. Barr
Keyword(s):  
Membrane Trafficking ◽  
Primary Cilia ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Cytoskeleton ◽  
Gtpase Activating Proteins ◽  
Cilia Formation ◽  
Sensory Structures ◽  
And Function ◽  
Specific Membrane

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

Plant cytokinesis: a tale of membrane traffic and fusion

2015 ◽  
Vol 43 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Gerd Jürgens ◽  
Misoon Park ◽  
Sandra Richter ◽  
Sonja Touihri ◽  
Cornelia Krause ◽  
...  
Keyword(s):  
Higher Plants ◽  
Membrane Vesicles ◽  
Cell Plate ◽  
Division Plane ◽  
Daughter Cells ◽  
Division Site ◽  
Plant Cytokinesis ◽  
M Phase ◽  
Golgi Network ◽  
Sm Protein

Cytokinesis separates the forming daughter cells. Higher plants have lost the ability to constrict the plasma membrane (PM) in the division plane. Instead, trans-Golgi network (TGN)-derived membrane vesicles are targeted to the centre of the division plane and generate, by homotypic fusion, the partitioning membrane named cell plate (CP). The CP expands in a centrifugal fashion until its margin fuses with the PM at the cortical division site. Mutant screens in Arabidopsis have identified a cytokinesis-specific syntaxin named KNOLLE and an interacting Sec1/Munc18 (SM) protein named KEULE both of which are required for vesicle fusion during cytokinesis. KNOLLE is only made during M-phase, targeted to the division plane and degraded in the vacuole at the end of cytokinesis. Here we address mechanisms of KNOLLE trafficking and interaction of KNOLLE with different soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) partners and with SM-protein KEULE, ensuring membrane fusion in cytokinesis.

scholarly journals B0AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Function ◽  
2021 ◽  
Author(s):  
Bruce R Stevens ◽  
J Clive Ellory ◽  
Robert L Preston
Keyword(s):  
Amino Acid ◽  
Membrane Trafficking ◽  
Functional Unit ◽  
Membrane Vesicles ◽  
High Energy ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Transport Activity ◽  
Acid Transporter

Abstract The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B0AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).

scholarly journals Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

2016 ◽  
Vol 27 (16) ◽  
pp. 2528-2541 ◽  
Author(s):  
Yajun Liu ◽  
I-Ju Lee ◽  
Mingzhai Sun ◽  
Casey A. Lower ◽  
Kurt W. Runge ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Cellular Localization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
The Novel ◽  
Septum Formation ◽  
Division Site ◽  
And Function

Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast.

scholarly journals The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00420-18 ◽  
Author(s):  
C. E. Melia ◽  
H. M. van der Schaar ◽  
A. W. M. de Jong ◽  
H. R. Lyoo ◽  
E. J. Snijder ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Encephalomyocarditis Virus ◽  
Escape Mutant ◽  
Membrane Structures ◽  
Double Membrane ◽  
Single Membrane ◽  
Phosphatidylinositol 4

ABSTRACTPicornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for theEnterovirusgenus of thePicornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of theCardiovirusgenus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCELike all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genusEnterovirus. The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro

Development ◽  
1988 ◽  
Vol 103 (3) ◽  
pp. 553-566 ◽  
Author(s):  
C.J. Hutchison ◽  
R. Cox ◽  
C.C. Ford
Keyword(s):  
Dna Synthesis ◽  
Chromatin Condensation ◽  
Membrane Vesicles ◽  
Pulse Labelling ◽  
Membrane Structures ◽  
Membrane Breakdown ◽  
A Cell ◽  
Chromosome Formation ◽  
Pronuclear Formation

Cell-free extracts prepared from Xenopus eggs support chromosome decondensation and pronuclear formation on demembranated sperm heads. 32P-dCTP pulse-labelling studies demonstrate that DNA synthesis occurs in multiple bursts of 30–40 min in extracts containing pronuclei, each burst being followed by a period of 20–50 min during which no synthesis occurs. Density substitution with bromodeoxyuridine indicates that the synthesis in each burst is semiconservative and results from new initiations, and that, following multiple bursts of synthesis, reinitiation events can occur. Changes in nuclear morphology have been characterized in the extract by phase-contrast microscopy and by fluorescence microscopy following pulse labelling with biotin-11-dUTP and staining with anti-lamin antibodies. Lamin accumulation occurs as DNA decondenses and parallels the acquisition of membrane structures. Biotin-11-dUTP incorporation is first observed in small nuclei having decondensed DNA and an extensive lamina. While DNA synthesis is occurring nuclei remain relatively small, but rapid swelling accompanied by chromosome condensation occurs when biotin incorporation ceases. Nuclear swelling and chromatin condensation is followed by nuclear membrane breakdown, lamin dispersal and chromosome formation. Mitosis lasts for approximately 20 min. Nuclear reassembly is recognized by the appearance of membrane vesicles around small pieces of decondensed DNA, which parallels the appearance of lamin islands within a chromatin mass. These ‘islands’ incorporate biotin, indicating that DNA synthesis is occurring, and apparently fuse as larger S-phase nuclei are formed. Extensive protein synthesis occurs for at least 4 h in most extracts. This synthesis is required for the initiation of mitotic events and the reinitiation of DNA synthesis.

Division Plane Establishment and Cytokinesis

2019 ◽  
Vol 70 (1) ◽  
pp. 239-267 ◽  
Author(s):  
Pantelis Livanos ◽  
Sabine Müller
Keyword(s):  
Secretory Pathway ◽  
Preprophase Band ◽  
Cell Plate ◽  
Division Plane ◽  
Spatial Reference ◽  
Division Site ◽  
Eukaryotic Proteins ◽  
Plate Formation ◽  
Membrane Compartment ◽  
Cytoplasmic Content

Plant cells divide their cytoplasmic content by forming a new membrane compartment, the cell plate, via a rerouting of the secretory pathway toward the division plane aided by a dynamic cytoskeletal apparatus known as the phragmoplast. The phragmoplast expands centrifugally and directs the cell plate to the preselected division site at the plasma membrane to fuse with the parental wall. The division site is transiently decorated by the cytoskeletal preprophase band in preprophase and prophase, whereas a number of proteins discovered over the last decade reside continuously at the division site and provide a lasting spatial reference for phragmoplast guidance. Recent studies of membrane fusion at the cell plate have revealed the contribution of functionally conserved eukaryotic proteins to distinct stages of cell plate biogenesis and emphasize the coupling of cell plate formation with phragmoplast expansion. Together with novel findings concerning preprophase band function and the setup of the division site, cytokinesis and its spatial control remain an open-ended field with outstanding and challenging questions to resolve.

scholarly journals Scw1p Antagonizes the Septation Initiation Network To Regulate Septum Formation and Cell Separation in the Fission Yeast Schizosaccharomyces pombe

2003 ◽  
Vol 2 (3) ◽  
pp. 510-520 ◽  
Author(s):  
Quan-Wen Jin ◽  
Dannel McCollum
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Separation ◽  
Deletion Mutant ◽  
Temperature Sensitive ◽  
Growth Defects ◽  
Septum Formation ◽  
Primary Septum ◽  
Septation Initiation Network ◽  
Ring Constriction

ABSTRACT Cytokinesis in the fission yeast Schizosaccharomyces pombe is regulated by a signaling pathway termed the septation initiation network (SIN). The SIN is essential for initiation of actomyosin ring constriction and septum formation. In a screen to search for mutations that can rescue the sid2-250 SIN mutant, we obtained scw1-18. Both the scw1-18 mutant and the scw1 deletion mutant (scw1Δ mutant), have defects in cell separation. Both the scw1-18 and scw1Δ mutations rescue the growth defects of not just the sid2-250 mutant but also the other temperature-sensitive SIN mutants. Other cytokinesis mutants, such as those defective for actomyosin ring formation, are not rescued by scw1Δ. scw1Δ does not seem to rescue the SIN by restoring SIN signaling defects. However, scw1Δ may function downstream of the SIN to promote septum formation, since scw1Δ can rescue the septum formation defects of the cps1-191β-1,3-glucan synthase mutant, which is required for synthesis of the primary septum.

scholarly journals The Actomyosin Ring Recruits Early Secretory Compartments to the Division Site in Fission Yeast

2008 ◽  
Vol 19 (3) ◽  
pp. 1125-1138 ◽  
Author(s):  
Aleksandar Vjestica ◽  
Xin-Zi Tang ◽  
Snezhana Oliferenko
Keyword(s):  
Endoplasmic Reticulum ◽  
Fission Yeast ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Physical Force ◽  
Daughter Cells ◽  
Division Site ◽  
Ring Assembly ◽  
Membrane Barrier ◽  
Ring Constriction

The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring.

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