scholarly journals Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth

2011 ◽  
Vol 22 (5) ◽  
pp. 624-633 ◽  
Author(s):  
Kelly Orlando ◽  
Xiaoli Sun ◽  
Jian Zhang ◽  
Tu Lu ◽  
Lauren Yokomizo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Growth ◽  
Membrane Trafficking ◽  
Daughter Cell ◽  
Lateral Diffusion ◽  
Cell Cortex ◽  
Actin Organization ◽  
Cell Plasma ◽  
Biochemical Fractionation ◽  
Asymmetric Growth

Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.

scholarly journals Interaction of Sla2p's ANTH Domain with PtdIns(4,5)P2 Is Important for Actin-dependent Endocytic Internalization

2005 ◽  
Vol 16 (2) ◽  
pp. 717-730 ◽  
Author(s):  
Yidi Sun ◽  
Marko Kaksonen ◽  
David T. Madden ◽  
Randy Schekman ◽  
David G. Drubin
Keyword(s):  
Cell Growth ◽  
Fluorescence Microscopy ◽  
Particle Tracking ◽  
Binding Assay ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Organization ◽  
Lysine Residues ◽  
Endocytic Machinery

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

scholarly journals Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth

2007 ◽  
Vol 176 (6) ◽  
pp. 771-777 ◽  
Author(s):  
Bing He ◽  
Fengong Xi ◽  
Jian Zhang ◽  
Daniel TerBush ◽  
Xiaoyu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Cell Growth ◽  
Daughter Cell ◽  
Protein Processing ◽  
Vesicle Formation ◽  
Secretory Vesicles ◽  
Early Stages ◽  
Polarized Cell Growth ◽  
Vesicle Secretion

In budding yeast, two classes of post-Golgi secretory vesicles carrying different sets of cargoes typified by Bgl2p and invertase are delivered to the plasma membrane for secretion. The exocyst is implicated in tethering these vesicles to the daughter cell membrane for exocytosis. In this study, we report that mutations in the exocyst component Exo70p predominantly block secretion of the Bgl2p vesicles. Furthermore, a defect in invertase vesicle trafficking caused by vps1Δ or pep12Δ in the exo70 mutant background is detrimental to the cell. The secretion defect in exo70 mutants was most pronounced during the early budding stage, which affected daughter cell growth. The selective secretion block does not occur at the vesicle formation or sorting stage because the exocytic vesicles are properly generated and protein processing is normal in the exo70 mutants. Our study suggests that Exo70p functions primarily at early stages of the cell cycle in Bgl2p vesicle secretion, which is critical for polarized cell growth.

scholarly journals Mesoscale Dynamics of Spectrin and Acto-Myosin shape Membrane Territories during Mechanoresponse

2019 ◽  
Author(s):  
Andrea Ghisleni ◽  
Camilla Galli ◽  
Pascale Monzo ◽  
Flora Ascione ◽  
Marc-Antoine Fardin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Membrane Trafficking ◽  
Cell Mechanics ◽  
Dynamic Interaction ◽  
The Other ◽  
Cell Cortex ◽  
Mesoscale Dynamics ◽  
Spectrin Cytoskeleton ◽  
Dynamic Interplay

AbstractThe spectrin cytoskeleton is a major component of the cell cortex. While ubiquitously expressed, its dynamic interaction with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, is poorly understood. Here, we investigated how the spectrin cytoskeleton re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We found spectrin and acto-myosin cytoskeletons to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence to organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin cytoskeletons necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.

scholarly journals A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast

2017 ◽  
Vol 28 (20) ◽  
pp. 2589-2599 ◽  
Author(s):  
Jesse Clarke ◽  
Noah Dephoure ◽  
Ira Horecka ◽  
Steven Gygi ◽  
Douglas Kellogg
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Cell Growth ◽  
Membrane Trafficking ◽  
Ribosome Biogenesis ◽  
Essential Role ◽  
Budding Yeast ◽  
Control Cell ◽  
Signaling Network ◽  
Membrane Growth

In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth-­dependent signals control cell growth and the cell cycle.

scholarly journals Diffusion and regionalization in membranes of maturing ram spermatozoa.

1984 ◽  
Vol 98 (5) ◽  
pp. 1678-1684 ◽  
Author(s):  
D E Wolf ◽  
J K Voglmayr
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Essential Feature ◽  
Lateral Diffusion ◽  
Mammalian Sperm ◽  
Epididymal Maturation ◽  
Cell Plasma ◽  
Sperm Plasma Membrane ◽  
Membrane Components

An essential feature of the "fluid mosaic model" (Singer, S. J., and G. L. Nicolson , 1972, Science (Wash. DC)., 175:720-731) of the cell plasma membrane is the ability of membrane lipids and proteins to diffuse laterally in the plane of the membrane. Mammalian sperm are capable of overcoming free random diffusion and restricting specific membrane components, both lipid and protein, to defined regions of the sperm's surface. The patterns of these regionalizations evolve with the processes of sperm differentiation: spermatogenesis, epididymal maturation, and capacitation. We have used the technique of fluorescence recovery after photobleaching to measure the diffusion of the lipid analogue 1,1'- dihexadecyl 3,3,3',3'- tetramethylindocarbocyanine perchlorate ( C16dil ) on the different morphological regions of testicular and ejaculated ram spermatozoa. We have found: (a) that the major morphologically distinct regions (head, midpiece, and tail) of the plasma membrane of both testicular and ejaculated spermatozoa are also physically distinct as measured by C16dil diffusibility; (b) that despite regional differences in diffusibility there is exchange of this lipid analogue by lateral diffusion between the major morphological regions of the plasma membrane; and (c) that epididymal maturation results in changes in C16dil diffusibility in the different regions of the sperm plasma membrane. In particular, the plasma membranes of the anterior and posterior heads become physically distinct.

scholarly journals Complementary mesoscale dynamics of spectrin and acto-myosin shape membrane territories during mechanoresponse

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrea Ghisleni ◽  
Camilla Galli ◽  
Pascale Monzo ◽  
Flora Ascione ◽  
Marc-Antoine Fardin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Membrane Trafficking ◽  
Cell Mechanics ◽  
Membrane Skeleton ◽  
The Other ◽  
Cell Cortex ◽  
Dynamic Interactions ◽  
Mesoscale Dynamics ◽  
Dynamic Interplay

Abstract The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.

scholarly journals Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells

2012 ◽  
Vol 303 (9) ◽  
pp. F1325-F1332 ◽  
Author(s):  
Hua Su ◽  
Conner B. Carter ◽  
Oskar Laur ◽  
Jeff M. Sands ◽  
Guangping Chen
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mdck Cells ◽  
Proteasome Inhibitors ◽  
Endocytic Pathway ◽  
Urea Transporter ◽  
Cell Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Cell Plasma ◽  
Darby Canine Kidney

The adenylyl cyclase stimulator forskolin (FSK) stimulates UT-A1 phosphorylation, membrane trafficking, and urea transport activity. Here, we found that FSK stimulation induces UT-A1 ubiquitination in UT-A1 Madin-Darby canine kidney (MDCK) cells. This suggests that phosphorylation by FSK also triggers the protein degradation machinery for UT-A1. UT-A1-MDCK cells were treated with 100 μg/ml cycloheximide to inhibit protein synthesis, with or without 10 μM FSK. Total UT-A1 protein abundance was significantly reduced after FSK treatment, concomitantly ubiquitinated UT-A1 was increased. We then specifically investigated the effect of FSK on UT-A1 expressed on the cell plasma membrane. FSK treatment accelerated UT-A1 removal from the cell plasma membrane by increasing UT-A1 endocytosis as judged by biotinylation/MesNa treatment and confocal microscopy. We further found that inhibition of the clathrin-mediated endocytic pathway, but not the caveolin-mediated endocytic pathway, significantly blocks FSK-stimulated UT-A1 endocytosis. The PKA inhibitor H89 and the proteasome inhibitors MG132 and lactacystin reduced FSK-induced membrane UT-A1 reduction. Our study shows that FSK activates the UT-A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation of UT-A1, which represents an important mechanism for the cell to return to the basal conditions after vasopressin stimulation.

scholarly journals A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast

2017 ◽  
Author(s):  
Jesse Clarke ◽  
Noah Dephoure ◽  
Ira Horecka ◽  
Steven Gygi ◽  
Douglas Kellogg
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Cell Growth ◽  
Membrane Trafficking ◽  
Ribosome Biogenesis ◽  
Essential Role ◽  
Budding Yeast ◽  
Control Cell ◽  
Signaling Network ◽  
Membrane Growth

AbstractIn budding yeast, cell cycle progression and ribosome biogenesis are dependent upon plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here, we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network plays an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together, these discoveries provide new clues to how growth-dependent signals control cell growth and the cell cycle.

Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties

1999 ◽  
Vol 5 (S2) ◽  
pp. 998-999
Author(s):  
Seung K. Rhee ◽  
Arjan P. Quist ◽  
Hai Lin ◽  
Nils Almqvist ◽  
Ratneshx Lai
Keyword(s):  
Plasma Membrane ◽  
Cell Volume ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Uptake ◽  
Aortic Endothelial Cells ◽  
Atomic Force ◽  
Cell Plasma ◽  
Uptake Assay ◽  
Gap Junctional

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

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