Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue

Developments in single-molecule microscopy (SMM) have enabled imaging individual proteins in biological systems, focusing on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied dynamics of the membrane protein H-Ras, its membrane-anchoring domain, C10H-Ras, and mutants, using total internal reflection fluorescence microscopy (TIRFM). Our results consistently confirm the presence of fast- and slow-diffusing subpopulations of molecules, which confine to microdomains within the plasma membrane. The active mutant H-RasV12 exhibits higher diffusion rates and is confined to larger domains than the wild-type H-Ras and its inactive mutant H-RasN17. Subsequently, we demonstrate that the structure and composition of the plasma membrane have an imperative role in modulating H-Ras mobility patterns. Ultimately, we establish that differences between cells within the same embryo largely contribute to the overall data variability. Our findings agree with a model where the cell architecture and the protein activation state determine protein mobility, underlining the importance of SMM imaging to study factors influencing protein dynamics in an intact living organism.

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Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue

10.1101/2021.06.21.449125 ◽

2021 ◽

Author(s):

Radoslaw J Gora ◽

Babette de Jong ◽

Patrick van Hage ◽

Mary Ann Rhiemus ◽

Fjodor van Steenis ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Dynamics ◽

Single Molecule ◽

Zebrafish Embryo ◽

Cultured Cells ◽

Living Organism ◽

Cellular Heterogeneity ◽

Mobility Patterns ◽

Membrane Anchoring

Over the last two decades, developments in single-molecule microscopy (SMM) have enabled imaging and tracking of individual, fluorescently labelled proteins in biological systems, and most of these studies have focused on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied the protein dynamics of the membrane protein H-Ras, mutants of this protein, and its membrane-anchoring domain, C10H-Ras, in epidermal cells of living two-day-old embryos, using a total internal reflection fluorescence microscopy (TIRFM) setup. For all proteins studied, our results consistently confirm the presence of a fast- and a slow-diffusing subpopulations of molecules, which both confine to microdomains within the plasma membrane. Although the mobility patterns of H-Ras, mutants of this proteins and its membrane-anchoring domain were remarkably similar, the constitutively active H-Ras mutant, H-RasV12, exhibited significantly higher diffusion rates than the wild-type H-Ras and its inactive mutant, H-RasN17. Ultimately, we characterized variability in our data obtained using the zebrafish embryo model and demonstrated that differences between cells within the same embryo are the largest source of variation in our data. Our findings are in line with a model in which the cellular architecture of individual cells within a tissue determine the mobility of H-Ras proteins anchored in the plasma membrane cytoplasmic leaflet. Thereby, our results underline the growing importance of performing SMM imaging in vivo in order to better understand factors influencing the protein dynamics in an intact living organism.

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Effects of cytoplasmic acidification on clathrin lattice morphology.

The Journal of Cell Biology ◽

10.1083/jcb.108.2.401 ◽

1989 ◽

Vol 108 (2) ◽

pp. 401-411 ◽

Cited By ~ 140

Author(s):

J Heuser

Keyword(s):

Plasma Membrane ◽

Cultured Cells ◽

Membrane Receptors ◽

The Other ◽

Culture Dish ◽

Initial Curvature ◽

Internal Ph ◽

Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

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Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

10.1101/2020.11.03.364851 ◽

2020 ◽

Author(s):

Javier Lizarrondo ◽

David P. Klebl ◽

Stephan Niebling ◽

Marc Abella ◽

Martin A. Schroer ◽

...

Keyword(s):

Plasma Membrane ◽

Complex Formation ◽

Dynamic Network ◽

Time Resolved ◽

Binding Domains ◽

Membrane Invagination ◽

Protein Membrane ◽

Membrane Anchoring

AbstractDuring clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites.

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Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

AJP Renal Physiology ◽

10.1152/ajprenal.00682.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. F935-F940 ◽

Cited By ~ 27

Author(s):

Janet D. Klein ◽

Mitsi A. Blount ◽

Otto Fröhlich ◽

Chad E. Denson ◽

Xiaoxiao Tan ◽

...

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Cell Lines ◽

Specific Antibody ◽

Cultured Cells ◽

Apical Plasma Membrane ◽

Wild Type ◽

Amino Acid Peptide ◽

In The Wild

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by 32P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

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Caveolae protect endothelial cells from membrane rupture during increased cardiac output

The Journal of Cell Biology ◽

10.1083/jcb.201504042 ◽

2015 ◽

Vol 211 (1) ◽

pp. 53-61 ◽

Cited By ~ 70

Author(s):

Jade P.X. Cheng ◽

Carolina Mendoza-Topaz ◽

Gillian Howard ◽

Jessica Chadwick ◽

Elena Shvets ◽

...

Keyword(s):

Endothelial Cells ◽

Cardiac Output ◽

Plasma Membrane ◽

Cultured Cells ◽

Protein Complexes ◽

Membrane Integrity ◽

Hemodynamic Forces ◽

Membrane Rupture ◽

Acute Rupture

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1−/− mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.

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In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

Nature Communications ◽

10.1038/ncomms13660 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 15

Author(s):

Adekunle T. Bademosi ◽

Elsa Lauwers ◽

Pranesh Padmanabhan ◽

Lorenzo Odierna ◽

Ye Jin Chai ◽

...

Keyword(s):

Single Molecule ◽

Neurotransmitter Release ◽

Motor Nerve ◽

Living Organism ◽

Neurosecretory Cells ◽

Single Particle Tracking ◽

Snare Complex ◽

Motor Nerve Terminal ◽

Activity Dependent

Abstract Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.

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Phenotypic heterogeneity in particle size is a viral mechanism of persistence

10.1101/843177 ◽

2019 ◽

Author(s):

Tian Li ◽

Zhenyu Li ◽

Erin E. Deans ◽

Eva Mittler ◽

Meisui Liu ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Ebola Virus ◽

Cultured Cells ◽

Functional Characterization ◽

Selective Advantage ◽

Protein Activation ◽

Virus Particles ◽

Fusion Inhibitors ◽

Functional Advantage

SUMMARYAssembly of many enveloped animal viruses yields a mixture of particle morphologies, from small, essentially isometric forms to vastly longer, filamentous forms. Selective advantage of pleomorphic virus structure is apparent only in vivo, hindering functional characterization of distinct particle shapes. Here we sought to mimic the in vivo pressures on virus entry in cultured cells and in single-particle experiments of membrane fusion for influenza virus preparations enriched in spherical or filamentous particles. We show that filamentous shape confers functional advantage in the presence of neutralizing antibodies or fusion inhibitors and in cases of only limited fusion-protein activation. For very long particles, inactivation of >95% of associated fusion proteins still permits enough active-protein cooperation to induce membrane merger. Experiments with Ebola virus-like particles show that resistance to antibody pressure is a conserved feature of filamentous particles. Our results offer a strategy for averting drug resistance or immune evasion by targeting filamentous virus particles.

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Deciphering dynamics of clathrin-mediated endocytosis in a living organism

The Journal of Cell Biology ◽

10.1083/jcb.201604128 ◽

2016 ◽

Vol 214 (3) ◽

pp. 347-358 ◽

Cited By ~ 28

Author(s):

Joshua P. Ferguson ◽

Nathan M. Willy ◽

Spencer P. Heidotting ◽

Scott D. Huber ◽

Matthew J. Webber ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Real Time ◽

Growth Rates ◽

Cultured Cells ◽

Living Organism ◽

Particle Detection ◽

Detection And Tracking ◽

Spatiotemporal Changes ◽

Multicellular Organisms

Current understanding of clathrin-mediated endocytosis (CME) dynamics is based on detection and tracking of fluorescently tagged clathrin coat components within cultured cells. Because of technical limitations inherent to detection and tracking of single fluorescent particles, CME dynamics is not characterized in vivo, so the effects of mechanical cues generated during development of multicellular organisms on formation and dissolution of clathrin-coated structures (CCSs) have not been directly observed. Here, we use growth rates of fluorescence signals obtained from short CCS intensity trace fragments to assess CME dynamics. This methodology does not rely on determining the complete lifespan of individual endocytic assemblies. Therefore, it allows for real-time monitoring of spatiotemporal changes in CME dynamics and is less prone to errors associated with particle detection and tracking. We validate the applicability of this approach to in vivo systems by demonstrating the reduction of CME dynamics during dorsal closure of Drosophila melanogaster embryos.

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Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

Nature Communications ◽

10.1038/s41467-021-23151-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Javier Lizarrondo ◽

David P. Klebl ◽

Stephan Niebling ◽

Marc Abella ◽

Martin A. Schroer ◽

...

Keyword(s):

Plasma Membrane ◽

Dynamic Network ◽

Stable Complex ◽

Time Resolved ◽

Binding Domains ◽

Membrane Invagination ◽

Scattering Study ◽

Membrane Anchoring

AbstractDuring clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.

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Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.957 ◽

2000 ◽

Vol 11 (3) ◽

pp. 957-968 ◽

Cited By ~ 49

Author(s):

Carol L. Manahan ◽

Madhavi Patnana ◽

Kendall J. Blumer ◽

Maurine E. Linder

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Yeast Cells ◽

Pheromone Response ◽

Guanine Nucleotide Binding Protein ◽

Α Subunit ◽

Protein Activation ◽

Pheromone Response Pathway ◽

G Protein Activation

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide–binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein γ subunit (Ste18p) is unusual among Gγsubunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the Gγsubunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of Gβγafter receptor-stimulated release from Gα. The G protein α subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

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