Staining of the Actin Cytoskeleton During Cell Division in Budding Yeast and Mammalian Cells

2022 ◽  
pp. 87-94
Author(s):  
Katie B. Shannon
Keyword(s):  
Cell Division ◽  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Budding Yeast

scholarly journals Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis

2011 ◽  
Vol 392 (8-9) ◽  
pp. 699-712 ◽  
Author(s):  
Michael A. McMurray ◽  
Christopher J. Stefan ◽  
Megan Wemmer ◽  
Greg Odorizzi ◽  
Scott D. Emr ◽  
...  
Keyword(s):  
Cell Division ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Early Stage ◽  
Cell Types ◽  
Cytoskeletal Proteins ◽  
Temperature Sensitive ◽  
Endosomal Sorting ◽  
Close Relatives

AbstractMembrane trafficking via targeted exocytosis to theSaccharomyces cerevisiaebud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects ofcla4Δ orelm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene,CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition inelm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.

scholarly journals Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

2005 ◽  
Vol 16 (1) ◽  
pp. 372-384 ◽  
Author(s):  
Avital A. Rodal ◽  
Lukasz Kozubowski ◽  
Bruce L. Goode ◽  
David G. Drubin ◽  
John H. Hartwig
Keyword(s):  
Cell Division ◽  
High Resolution ◽  
Actin Cytoskeleton ◽  
Yeast Cell ◽  
Budding Yeast ◽  
Model Organism ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Actin Patch ◽  
Order Structures

Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs

1996 ◽  
Vol 109 (2) ◽  
pp. 367-377 ◽  
Author(s):  
H. Dutartre ◽  
J. Davoust ◽  
J.P. Gorvel ◽  
P. Chavrier
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Rho Gtpase ◽  
Laser Scanning Microscopy ◽  
Regulatory Subunit ◽  
Confocal Laser ◽  
Complex Architectures ◽  
Immunofluorescence Labelling

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

scholarly journals Pushing for answers: is myosin V directly involved in moving mitochondria?

2008 ◽  
Vol 181 (1) ◽  
pp. 15-18 ◽  
Author(s):  
Rajeshwari R. Valiathan ◽  
Lois S. Weisman
Keyword(s):  
Cell Division ◽  
Light Chain ◽  
Budding Yeast ◽  
Myosin V ◽  
Class V ◽  
Mitochondrial Transport ◽  
Direct Involvement ◽  
Cell Biol ◽  
Myosin Motors

In budding yeast, the actin-based class V myosin motors, Myo2 and Myo4, transport virtually all organelles from mother to bud during cell division. Until recently, it appeared that mitochondria may be an exception, with studies showing that the Arp2/3 complex is required for their movement. However, several recent studies have proposed that Myo2 has a direct involvement in mitochondria inheritance. In this issue, Altmann et al. (Altmann, K., M. Frank, D. Neumann, S. Jakobs, and B. Westermann. 2008. J. Cell Biol. 181:119–130) provide the strongest support yet that Myo2 and its associated light chain Mlc1 function directly and significantly in both mitochondria–actin interactions and in the movement of mitochondria from mother to bud. The conflicting functions of Arp 2/3 and Myo2 may be reconciled by the existence of multiple pathways involved in mitochondrial transport.

Overexpression Phenotypes of Plk1 and Ndrg2 in Schizosaccharomyces Pombe: Fission Yeast System for Mammalian Gene Study

2005 ◽  
Vol 277-279 ◽  
pp. 1-6 ◽  
Author(s):  
Young Joo Jang ◽  
Young Sook Kil ◽  
Jee Hee Ahn ◽  
Jae Hoon Ji ◽  
Jong Seok Lim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Genetic System ◽  
Functional Study ◽  
Mammalian Genes

The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

The structural mechanics of cell division in Trypanosoma brucei

2008 ◽  
Vol 36 (3) ◽  
pp. 421-424 ◽  
Author(s):  
Sue Vaughan ◽  
Keith Gull
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Single Copy ◽  
Structural Mechanics ◽  
Sub Saharan Africa ◽  
Mammalian Host

Undoubtedly, there are fundamental processes driving the structural mechanics of cell division in eukaryotic organisms that have been conserved throughout evolution and are being revealed by studies on organisms such as yeast and mammalian cells. Precision of structural mechanics of cytokinesis is however probably no better illustrated than in the protozoa. A dramatic example of this is the protozoan parasite Trypanosoma brucei, a unicellular flagellated parasite that causes a devastating disease (African sleeping sickness) across Sub-Saharan Africa in both man and animals. As trypanosomes migrate between and within a mammalian host and the tsetse vector, there are periods of cell proliferation and cell differentiation involving at least five morphologically distinct cell types. Much of the existing cytoskeleton remains intact during these processes, necessitating a very precise temporal and spatial duplication and segregation of the many single-copy organelles. This structural precision is aiding progress in understanding these processes as we apply the excellent reverse genetics and post-genomic technologies available in this system. Here we outline our current understanding of some of the structural aspects of cell division in this fascinating organism.

Nocodazole and cytochalasin D induce tetraploidy in mammalian cells

1984 ◽  
Vol 246 (1) ◽  
pp. C154-C156 ◽  
Author(s):  
G. W. Zieve
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Cytochalasin D ◽  
Microtubule Assembly ◽  
Reversible Inhibitor ◽  
Cleavage Furrow ◽  
Synchronized Cells ◽  
Cells In Culture ◽  
Wide Range

Nocodazole, a rapidly reversible inhibitor of microtubule assembly is useful for preparing mammalian cells synchronized at all stages of mitosis. When synchronized cells are allowed to progress through mitosis in the presence of cytochalasin D, the cleavage furrow is inhibited and dikaryon cells are formed. These cells become homogeneous populations of stable mononuclear tetraploid cells after the following cell division. This procedure is applicable to a wide range of mammalian cells in culture.

scholarly journals Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

2021 ◽  
pp. 75-118
Author(s):  
Sarah Lecinski ◽  
Jack W. Shepherd ◽  
Lewis Frame ◽  
Imogen Hayton ◽  
Chris MacDonald ◽  
...  
Keyword(s):  
Cell Division ◽  
Budding Yeast ◽  
Hyperosmotic Stress ◽  
Molecular Crowding
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