F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

We have visualised F-actin and microtubules in living Tradescantia virginiana stamen hair cells by confocal laser scanning microscopy after microinjecting rhodamine-phalloidin or carboxyfluorescein-labelled brain tubulin. We monitored these components of the cytoskeleton as the cells prepared for division at preprophase and progressed through mitosis to cytokinesis. Reorganisation of the interphase cortical cytoskeleton results in preprophase bands of both F-actin and microtubules that coexist in the cell cortex, centred on the site at which the future cell plate will fuse with the parent cell wall. The preprophase band of microtubules is formed from microtubules that polymerise and incorporate tubulin during prophase. The preprophase band of actin may form either by reorganisation of pre-existing filaments or by de novo polymerisation. Both cytoskeletal components disappear from the future division site approximately five minutes prior to the breakdown of the nuclear envelope. Cortical microtubules are undetectable throughout mitosis and cytokinesis, whereas cortical F-actin remains abundant, although it is notably excluded from the division site. The phragmoplast, containing both F-actin and microtubules, expands towards the cortical actin exclusion-zone through a region that has no detectable microtubules or F-actin. The phragmoplast comes to rest in the predefined region of the cortex that is devoid of F-actin. It is proposed that cortical F-actin may act as a “negative” template which could position the phragmoplast and cell plate correctly. This is the first in vivo documentation of F- actin dynamics at the division site in living plant cells.

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Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146497 ◽

1993 ◽

Vol 51 ◽

pp. 130-131

Author(s):

Ann Cleary

Keyword(s):

Cell Division ◽

Fluorescent Probes ◽

Actin Filaments ◽

Intracellular Transport ◽

Cell Structure ◽

Plant Cells ◽

Cell Plate ◽

Cell Cortex ◽

Living Plant ◽

Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

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Faculty Opinions recommendation of The Min system and nucleoid occlusion are not required for identifying the division site in Bacillus subtilis but ensure its efficient utilization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.14263040.15775167 ◽

2012 ◽

Author(s):

Petra Levin

Keyword(s):

Bacillus Subtilis ◽

Efficient Utilization ◽

Division Site

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Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA

International Journal of Molecular Sciences ◽

10.3390/ijms22158350 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8350

Author(s):

Naďa Labajová ◽

Natalia Baranova ◽

Miroslav Jurásek ◽

Robert Vácha ◽

Martin Loose ◽

...

Keyword(s):

Cell Division ◽

Lipid Bilayers ◽

Bacterial Cell ◽

Lipid Membranes ◽

Model Organism ◽

Active Role ◽

Membrane Curvature ◽

Bacterial Cell Division ◽

Division Site ◽

Clostridioides Difficile

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.

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Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524538113 ◽

2016 ◽

Vol 113 (17) ◽

pp. 4788-4793 ◽

Cited By ~ 21

Author(s):

Monica Markovski ◽

Jessica L. Bohrhunter ◽

Tania J. Lupoli ◽

Tsuyoshi Uehara ◽

Suzanne Walker ◽

...

Keyword(s):

Polymerase Activity ◽

Activation Mechanism ◽

Phenotypic Analysis ◽

Outer Membranes ◽

Division Site ◽

Physiological Relevance ◽

Membrane Lipoproteins ◽

Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

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Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e16-03-0156 ◽

2016 ◽

Vol 27 (16) ◽

pp. 2528-2541 ◽

Cited By ~ 6

Author(s):

Yajun Liu ◽

I-Ju Lee ◽

Mingzhai Sun ◽

Casey A. Lower ◽

Kurt W. Runge ◽

...

Keyword(s):

Fission Yeast ◽

Rho Gtpases ◽

Cellular Localization ◽

Affinity Purification ◽

Coiled Coil ◽

The Novel ◽

Septum Formation ◽

Division Site ◽

And Function

Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast.

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Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

The Journal of Cell Biology ◽

10.1083/jcb.118.3.561 ◽

1992 ◽

Vol 118 (3) ◽

pp. 561-571 ◽

Cited By ~ 157

Author(s):

S Chowdhury ◽

K W Smith ◽

M C Gustin

Keyword(s):

Osmotic Stress ◽

Actin Filament ◽

Physiological Process ◽

Actin Binding ◽

Temperature Sensitive ◽

Filamentous Actin ◽

Cortical Actin ◽

Yeast Saccharomyces Cerevisiae ◽

Rhodamine Phalloidin ◽

Diploid Cells

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

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Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin.

The Journal of Cell Biology ◽

10.1083/jcb.113.5.1057 ◽

1991 ◽

Vol 113 (5) ◽

pp. 1057-1067 ◽

Cited By ~ 149

Author(s):

M L Vitale ◽

A Rodríguez Del Castillo ◽

L Tchakarov ◽

J M Trifaró

Keyword(s):

Actin Filament ◽

Chromaffin Cells ◽

Chromaffin Cell ◽

Catecholamine Secretion ◽

Cortical Region ◽

Cortical Surface ◽

Filamentous Actin ◽

Cell Stimulation ◽

Rhodamine Phalloidin ◽

Filament Networks

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

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Purification and characterization of a nonpolymerizing long-pitch actin dimer

Biochemistry and Cell Biology ◽

10.1139/o06-091 ◽

2006 ◽

Vol 84 (5) ◽

pp. 695-702 ◽

Cited By ~ 1

Author(s):

Braden Sweeting ◽

John F. Dawson

Keyword(s):

Critical Concentration ◽

Dnase I ◽

Actin Binding ◽

Wild Type ◽

Filamentous Actin ◽

Rhodamine Phalloidin ◽

Fold Reduction ◽

Crystallization Conditions ◽

Self Association ◽

Pointed End

Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin. The Q41C/C374A mutant actin possessed altered polymerization properties, with a 2-fold reduction in the rate of elongation and an increased critical concentration relative to wild-type actin. The Q41C/C374A mutant actin also displayed an increase in the IC50 for DNase I, a pointed-end actin-binding protein. The long-pitch dimer was bound by DNase I to prevent polymerization and purified. It was found that each actin dimer is bound by 2 DNase I molecules, 1 likely bound to each of the actin protomers. The long-pitch dimer bound by DNase I did not form short F actin structures, as assessed by the binding of rhodamine–phalloidin.

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