scholarly journals Arabidopsis Myosin XIK Interacts with the Exocyst Complex to Facilitate Vesicle Tethering during Exocytosis

2020 ◽  
Author(s):  
Weiwei Zhang ◽  
Lei Huang ◽  
Chunhua Zhang ◽  
Christopher J. Staiger
Keyword(s):  
Mammalian Cells ◽  
Secretory Vesicle ◽  
Tail Domain ◽  
Spatiotemporal Resolution ◽  
Dynamic Regulation ◽  
Vesicle Tethering ◽  
Exocyst Complex ◽  
Myosin Xi ◽  
Myosin Motors

ABSTRACTMyosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely-growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to the SEC5B subunit of exocyst in vitro and functional fluorescently-tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the frequency and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes (CSCs) with high spatiotemporal resolution imaging and pair-wise colocalization analysis of myosin XIK, exocyst subunits and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the recruitment of exocyst to the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides new insights about the dynamic regulation of exocytosis in plants.

scholarly journals Revising the Role of Cortical Cytoskeleton During Secretion: Actin and Myosin XI Function in Vesicle Tethering

2021 ◽  
Author(s):  
Weiwei Zhang ◽  
Christopher J. Staiger
Keyword(s):  
Single Particle ◽  
Single Particle Tracking ◽  
Quantitative Approach ◽  
Spatiotemporal Resolution ◽  
Microtubule Inhibitor ◽  
Vesicle Tethering ◽  
Molecular Events ◽  
Myosin Xi ◽  
Cortical Cytoskeleton

AbstractIn plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase of transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.

scholarly journals Cardiac optogenetics

2013 ◽  
Vol 304 (9) ◽  
pp. H1179-H1191 ◽  
Author(s):  
Emilia Entcheva
Keyword(s):  
Ion Channels ◽  
Mammalian Cells ◽  
High Specificity ◽  
Emerging Technology ◽  
Inhibitory Response ◽  
Research Activity ◽  
Spatiotemporal Resolution ◽  
And Control ◽  
The Impact

Optogenetics is an emerging technology for optical interrogation and control of biological function with high specificity and high spatiotemporal resolution. Mammalian cells and tissues can be sensitized to respond to light by a relatively simple and well-tolerated genetic modification using microbial opsins (light-gated ion channels and pumps). These can achieve fast and specific excitatory or inhibitory response, offering distinct advantages over traditional pharmacological or electrical means of perturbation. Since the first demonstrations of utility in mammalian cells (neurons) in 2005, optogenetics has spurred immense research activity and has inspired numerous applications for dissection of neural circuitry and understanding of brain function in health and disease, applications ranging from in vitro to work in behaving animals. Only recently (since 2010), the field has extended to cardiac applications with less than a dozen publications to date. In consideration of the early phase of work on cardiac optogenetics and the impact of the technique in understanding another excitable tissue, the brain, this review is largely a perspective of possibilities in the heart. It covers the basic principles of operation of light-sensitive ion channels and pumps, the available tools and ongoing efforts in optimizing them, overview of neuroscience use, as well as cardiac-specific questions of implementation and ideas for best use of this emerging technology in the heart.

scholarly journals Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Marvin E Tanenbaum ◽  
Ronald D Vale ◽  
Richard J McKenney
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Motor Proteins ◽  
Cytoplasmic Dynein ◽  
Eukaryotic Cells ◽  
Reverse Direction ◽  
Tail Domain ◽  
Mitotic Spindles

Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins.

scholarly journals Revising the Role of Cortical Cytoskeleton during Secretion: Actin and Myosin XI Function in Vesicle Tethering

2021 ◽  
Vol 23 (1) ◽  
pp. 317
Author(s):  
Weiwei Zhang ◽  
Christopher J. Staiger
Keyword(s):  
Single Particle ◽  
Single Particle Tracking ◽  
Quantitative Approach ◽  
Spatiotemporal Resolution ◽  
Microtubule Inhibitor ◽  
Vesicle Tethering ◽  
Molecular Events ◽  
Myosin Xi ◽  
Cortical Cytoskeleton

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.

Human α-synemin interacts directly with vinculin and metavinculin

2008 ◽  
Vol 409 (3) ◽  
pp. 657-667 ◽  
Author(s):  
Ning Sun ◽  
David R. Critchley ◽  
Denise Paulin ◽  
Zhenlin Li ◽  
Richard M. Robson
Keyword(s):  
Mammalian Cells ◽  
Striated Muscle ◽  
Splice Variants ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Protein Protein Interaction

Synemin is a very large, unique member of the IF (intermediate filament) protein superfamily. Association of synemin with the major IF proteins, desmin and/or vimentin, within muscle cells forms heteropolymeric IFs. We have previously identified interactions of avian synemin with α-actinin and vinculin. Avian synemin, however, is expressed as only one form, whereas human synemin is expressed as two major splice variants, namely α- and β-synemins. The larger α-synemin contains an additional 312-amino-acid insert (termed SNTIII) located near the end of the long C-terminal tail domain. Whether α- and β-synemins have different cellular functions is unclear. In the present study we show, by in vitro protein–protein interaction assays, that SNTIII interacts directly with both vinculin and metavinculin. Furthermore, SNTIII interacts with vinculin in vivo, and this association is promoted by PtdIns(4,5)P2. SNTIII also specifically co-localizes with vinculin within focal adhesions when transiently expressed in mammalian cells. In contrast, other regions of synemin show distinct localization patterns in comparison with those of SNTIII, without labelling focal adhesions. Our results indicate that α-synemin, but not β-synemin, interacts with both vinculin and metavinculin, thereby linking the heteropolymeric IFs to adhesion-type junctions, such as the costameres located within human striated muscle cells.

scholarly journals Autoregulation and Dual Stepping Mode of MYA2, an Arabidopsis Myosin XI Responsible for Cytoplasmic Streaming

2021 ◽  
Author(s):  
Takashi Haraguchi ◽  
Kohji Ito ◽  
Takamitsu Morikawa ◽  
Nao Shoji ◽  
Mitsuhiro Iwaki ◽  
...  
Keyword(s):  
Cytoplasmic Streaming ◽  
Force Measurement ◽  
Plant Cells ◽  
Transfer System ◽  
Tail Domain ◽  
Animal Cells ◽  
Myosin V ◽  
Myosin Xi ◽  
Stall Force

Abstract Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD), When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining TIRF microscopy and the FIONA method, revealed that the MYA2 processively moved on actin with three different step sizes: −28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes, hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2 − 3 pN). These results indicated that MYA2 is more flexible than the myosin V responsible for vesicle transport in animal cells. Such flexibility may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

scholarly journals mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

2006 ◽  
Vol 174 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Sidney Yu ◽  
Ayano Satoh ◽  
Marc Pypaert ◽  
Karl Mullen ◽  
Jesse C. Hay ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Tethering ◽  
Transitional Endoplasmic Reticulum ◽  
Large Complex ◽  
Copii Vesicle ◽  
Copii Vesicles

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

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