scholarly journals Spatial analysis of Cdc42 activity reveals a role for plasma membrane–associated Cdc42 in centrosome regulation

2017 ◽  
Vol 28 (15) ◽  
pp. 2135-2145 ◽  
Author(s):  
Kari A. Herrington ◽  
Andrew L. Trinh ◽  
Carolyn Dang ◽  
Ellen O’Shaughnessy ◽  
Klaus M. Hahn ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Spatial Analysis ◽  
Substantial Reduction ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Spatial Control ◽  
Cytoskeletal Organization ◽  
Cellular Processes ◽  
Phasor Analysis

The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by moni­toring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase.

scholarly journals CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, β1 integrin recycling, and cell migration

2019 ◽  
Vol 12 (579) ◽  
pp. eaav5938 ◽  
Author(s):  
Mallika Ghosh ◽  
Robin Lo ◽  
Ivan Ivic ◽  
Brian Aguilera ◽  
Veneta Qendro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Attachment ◽  
Leading Edge ◽  
Small Gtpase ◽  
Β1 Integrin ◽  
Recycling Endosomes ◽  
Cellular Processes ◽  
Early Endosomes

Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the β1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with β1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized β1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.

scholarly journals Systematic spatial mapping of proteins at exocytic and endocytic structures

2014 ◽  
Vol 25 (13) ◽  
pp. 2084-2093 ◽  
Author(s):  
Ben T. Larson ◽  
Kem A. Sochacki ◽  
Jonathan M. Kindem ◽  
Justin W. Taraska
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Steady State ◽  
Pc12 Cells ◽  
Spatial Organization ◽  
Molecular Composition ◽  
Spatial Mapping ◽  
Cellular Processes ◽  
Single Frame ◽  
Vesicular Secretion

Vesicular secretion (exocytosis) involves the release and then compensatory recycling of vesicle components through endocytosis. This fundamental cellular process is controlled by the coordinated assembly and interactions of dozens of proteins at the plasma membrane. Understanding the molecular composition of individual exocytic and endocytic structures and their organization across the plasma membrane is critical to understanding the behavior and regulation of these two cellular processes. Here we develop a high-resolution and high-throughput fluorescence imaging–based approach for the unbiased mapping of 78 proteins at single exocytic vesicles and endocytic structures in neuroendocrine PC12 cells. This analysis uses two-color single-frame images to provide a systems-level map of the steady-state distributions of proteins at individual exocytic and endocytic structures in the cell. Along with this quantitative map, we find that both calcium-regulated exocytic vesicles (dense core vesicles) and endocytic structures (clathrin-coated structures) and the proteins associated with these structures exhibit a random spatial distribution in unstimulated neuroendocrine PC12 cells. This approach is broadly applicable for quantitatively mapping the molecular composition and spatial organization of discrete cellular processes with central molecular hubs.

scholarly journals RAB11-Mediated Trafficking and Human Cancers: An Updated Review

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 26
Author(s):  
Elsi Ferro ◽  
Carla Bosia ◽  
Carlo C. Campa
Keyword(s):  
Plasma Membrane ◽  
Normal Cell ◽  
Vesicular Trafficking ◽  
Small Gtpase ◽  
Cell Physiology ◽  
Biological Mechanisms ◽  
Cellular Processes ◽  
Human Cancers ◽  
Toxic Materials ◽  
Dependent Pathway

Many disorders block and subvert basic cellular processes in order to boost their progression. One protein family that is prone to be altered in human cancers is the small GTPase RAB11 family, the master regulator of vesicular trafficking. RAB11 isoforms function as membrane organizers connecting the transport of cargoes towards the plasma membrane with the assembly of autophagic precursors and the generation of cellular protrusions. These processes dramatically impact normal cell physiology and their alteration significantly affects the survival, progression and metastatization as well as the accumulation of toxic materials of cancer cells. In this review, we discuss biological mechanisms ensuring cargo recognition and sorting through a RAB11-dependent pathway, a prerequisite to understand the effect of RAB11 alterations in human cancers.

scholarly journals Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum

Development ◽  
2001 ◽  
Vol 128 (16) ◽  
pp. 3133-3144 ◽  
Author(s):  
Takao Yamasaki ◽  
Kousuke Kawaji ◽  
Katsuhiko Ono ◽  
Haruhiko Bito ◽  
Tomoo Hirano ◽  
...  
Keyword(s):  
Granule Cell ◽  
Molecular Mechanisms ◽  
Granule Cells ◽  
Ectopic Expression ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Fiber Formation ◽  
Parallel Fiber ◽  
Differentiation Markers ◽  
Cytoskeletal Organization

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats(rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics byPax6 is independent of the ROCK-mediated Rho small GTPase pathway. We propose that in addition to its roles during early patterning of the CNS,Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons.

scholarly journals Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase

2019 ◽  
Author(s):  
Iker Lamas ◽  
Laura Merlini ◽  
Aleksandar Vještica ◽  
Vincent Vincenzetti ◽  
Sophie G Martin
Keyword(s):  
Plasma Membrane ◽  
Positive Feedback ◽  
Active Zone ◽  
Feedback Regulation ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Yeast Cells ◽  
Ras Gtpase ◽  
Dependent Protein ◽  
Protein Recruitment

AbstractThe small GTPase Cdc42 is critical for cell polarization. Scaffold-mediated positive feedback regulation was proposed to mediate symmetry-breaking to a single active zone in budding yeast cells. In rod-shaped fission yeast S. pombe cells, active Cdc42-GTP localizes to both cell poles, where it promotes bipolar growth. Here, we implement the CRY2-CIBN optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the GEF Scd1, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is completely bypassed and positive feedback restored by an engineered interaction between the GEF and a Cdc42 effector, the Pak1 kinase. Remarkably, such re-wired cells are viable and grow in a bipolar manner even when lacking otherwise essential Cdc42 activators. Interestingly, these cells reveal that Ras1 GTPase plays a dual role in localizing and activating the GEF, thus potentiating the feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, is minimally required for rod-shape formation.

scholarly journals A Two-Tiered Mechanism Enables Localized Cdc42 Signaling during Enterocyte Polarization

2017 ◽  
Vol 37 (7) ◽  
Author(s):  
Lucas J. M. Bruurs ◽  
Susan Zwakenberg ◽  
Mirjam C. van der Net ◽  
Fried J. Zwartkruis ◽  
Johannes L. Bos
Keyword(s):  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Differential Diffusion ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Dual Mechanism

ABSTRACT Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

vitreal cells and specialized phagocytes in the chick embryo retina: A TEM and SEM study

1990 ◽  
Vol 48 (3) ◽  
pp. 330-331
Author(s):  
M.A. Cuadros ◽  
M.J. Martinez-Guerrero ◽  
A. Rios
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Sem Images ◽  
Intimate Contact ◽  
Cellular Processes ◽  
Sem Study ◽  
Free Cells

In the chick embryo retina (days 3-4 of incubation), coinciding with an increase in cell death, specialized phagocytes characterized by intense acid phosphatase activity have been described. In these preparations, all free cells in the vitreal humor (vitreal cells) were strongly labeled. Conventional TEM and SEM techniques were used to characterize them and attempt to determine their relationship with retinal phagocytes.Two types of vitreal cells were distinguished. The first are located at some distance from the basement membrane of the neuroepithelium, and are rounded, with numerous vacuoles and thin cytoplasmic prolongations. Images of exo- and or endocytosis were frequent; the cells showed a well-developed Golgi apparatus (Fig. 1) In SEM images, the cells was covered with short cellular processes (Fig. 3). Cells lying parallel to or alongside the basement membrane are elongated. The plasma membrane is frequently in intimate contact with the basement membrane. These cells have generally a large cytoplasmic expansion (Fig. 5).

scholarly journals Spatial Analysis (Measurements at Heights of 10 m and 20 m above Ground Level) of the Concentrations of Particulate Matter (PM10, PM2.5, and PM1.0) and Gaseous Pollutants (H2S) on the University Campus: A Case Study

Atmosphere ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 62
Author(s):  
Robert Cichowicz ◽  
Maciej Dobrzański
Keyword(s):  
Spatial Distribution ◽  
Hydrogen Sulfide ◽  
Particulate Matter ◽  
Air Quality ◽  
Spatial Analysis ◽  
Ground Level ◽  
Leeward Side ◽  
High Concentration ◽  
Above Ground Level ◽  
High Concentrations

Spatial analysis of the distribution of particulate matter PM10, PM2.5, PM1.0, and hydrogen sulfide (H2S) gas pollution was performed in the area around a university library building. The reasons for the subject matter were reports related to the perceptible odor characteristic of hydrogen sulfide and a general poor assessment of air quality by employees and students. Due to the area of analysis, it was decided to perform measurements at two heights, 10 m and 20 m above ground level, using measuring equipment attached to a DJI Matrice 600 unmanned aerial vehicle (UAV). The aim of the measurements was air quality assessment and investigate the convergence of the theory of air flow around the building with the spatial distribution of air pollutants. Considerable differences of up to 63% were observed in the concentrations of pollutants measured around the building, especially between opposite sides, depending on the direction of the wind. To explain these differences, the theory of aerodynamics was applied to visualize the probable airflow in the direction of the wind. A strong convergence was observed between the aerodynamic model and the spatial distribution of pollutants. This was evidenced by the high concentrations of dust in the areas of strong turbulence at the edges of the building and on the leeward side. The accumulation of pollutants was also clearly noticeable in these locations. A high concentration of H2S was recorded around the library building on the side of the car park. On the other hand, the air turbulence around the building dispersed the gas pollution, causing the concentration of H2S to drop on the leeward side. It was confirmed that in some analyzed areas the permissible concentration of H2S was exceeded.

scholarly journals Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2089 ◽  
Author(s):  
Iker Lamas ◽  
Nathalie Weber ◽  
Sophie G. Martin
Keyword(s):  
Fission Yeast ◽  
Positive Feedback ◽  
Antagonistic Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Architecture

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

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