Rhodamine-labelled phalloidin stains components in the chromosomal spindle fibres of crane-fly spermatocytes and Haemanthus endosperm cells

1992 ◽  
Vol 70 (8) ◽  
pp. 664-676 ◽  
Author(s):  
B. Barbara Czaban ◽  
Arthur Forer
Keyword(s):  
Cell Division ◽  
Actin Filaments ◽  
Spindle Fibres

In crane-fly spermatocytes and Haemanthus endosperm, all metaphase and anaphase chromosomal spindle fibres were stained with rhodamine-labelled phalloidin. In crane-fly spermatocytes, each kinetochore was stained with rhodamine-labelled phalloidin at diakinesis of prophase and after colcemid caused metaphase spindles to depolymerize. Since phalloidin stains actin filaments, the distributions of rhodamine-labelled phalloidin-stained material in crane-fly spermatocytes and Haemanthus endosperm suggest that actin filaments might interact with microtubules to produce forces that move chromosomes during cell division, either directly or via an intermediate motor molecule.Key words: phalloidin, chromosomal spindle fibres, kinetochores.

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

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.

scholarly journals Myosin motors fragment and compact membrane-bound actin filaments

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Sven K Vogel ◽  
Zdenek Petrasek ◽  
Fabian Heinemann ◽  
Petra Schwille
Keyword(s):  
Cell Division ◽  
Compressive Stress ◽  
Actin Filaments ◽  
Cell Cortex ◽  
Tirf Microscopy ◽  
Actin Cortex ◽  
Actin Turnover ◽  
Membrane Bound ◽  
Myosin Motors

Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis.

scholarly journals Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins

2017 ◽  
Author(s):  
Lam T. Nguyen ◽  
Matthew T. Swulius ◽  
Samya Aich ◽  
Mithilesh Mishra ◽  
Grant J. Jensen
Keyword(s):  
Experimental Data ◽  
Cell Division ◽  
Fluorescence Microscopy ◽  
Actin Filaments ◽  
Coarse Grained ◽  
Eukaryotic Cells ◽  
Ring Assembly ◽  
Assembly Result ◽  
Coarse Grained Simulations ◽  
Electron Cryotomography

AbstractCytokinesis in most eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by existing literature and new 3D molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin crosslinkers, and membranes and simulate their nteractions. Exploring a matrix of possible actomyosin configurations suggested that node-based architectures ike those presently described for ring assembly result in membrane puckers not seen in EM images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly-distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses.Significance StatementIn most eukaryotes, a ring of actin and myosin drives cell division, but how the elements of the ring are arranged and constrict remain unclear. Here we use 3D coarse-grained simulations to explore various possibilities. Our simulations suggest that if actomyosin is arranged in nodes (as suggested by a popular model of ring assembly), the membrane distorts in ways not seen experimentally. Instead, actin and myosin are more ikely uniformly distributed around the ring. In the model that best fits experimental data, ring tension is generated by interactions between bipolar myosins and actin, and transmitted to the membrane via unipolar myosins. Technologically the study highlights how coarse-grained simulations can test specific mechanistic hypotheses by comparing their predicted outcomes to experimental results.

Function of spindle microtubules in directing cortical movement and actin filament organization in dividing cultured cells

1996 ◽  
Vol 109 (8) ◽  
pp. 2041-2051 ◽  
Author(s):  
D.J. Fishkind ◽  
J.D. Silverman ◽  
Y.L. Wang
Keyword(s):  
Cell Division ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Linear Dichroism ◽  
Single Particle Tracking ◽  
Contractile Apparatus ◽  
Cortical Actin ◽  
Surface Receptors ◽  
Spindle Microtubules

The mitotic spindle has long been recognized to play an essential role in determining the position of the cleavage furrow during cell division, however little is known about the mechanisms involved in this process. One attractive hypothesis is that signals from the spindle may function to induce reorganization of cortical structures and transport of actin filaments to the equator during cytokinesis. While an important idea, few experiments have directly tested this model. In the present study, we have used a variety of experimental approaches to identify microtubule-dependent effects on key cortical events during normal cell cleavage, including cortical flow, reorientation of actin filaments, and formation of the contractile apparatus. Single-particle tracking experiments showed that the microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following anaphase onset, while the microtubule stabilizing drug taxol causes profound changes in the overall pattern of receptor movements. These effects were accompanied by a related set of changes in the organization of the actin cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less ordered than in controls. Measurements with fluorescence-detected linear dichroism indicated a decrease in the alignment of filaments along the spindle axis. In contrast, actin filaments in taxol-treated cells showed an increased alignment along the equator on both the ventral and dorsal cortical surfaces, mirroring the redistribution pattern of surface receptors. Together, these experiments show that spindle microtubules are involved in directing bipolar flow of surface receptors and reorganization of actin filaments during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the contractile apparatus of dividing tissue culture cells.

scholarly journals Anillin propels myosin-independent constriction of actin rings

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ondřej Kučera ◽  
Valerie Siahaan ◽  
Daniel Janda ◽  
Sietske H. Dijkstra ◽  
Eliška Pilátová ◽  
...  
Keyword(s):  
Cell Division ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Mechanical Forces ◽  
Actin Networks ◽  
Essential Step ◽  
Circular Structure ◽  
Ring Constriction ◽  
Contractile Forces ◽  
Actin Rings

AbstractConstriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that anillin, a non­motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. Our results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring.

scholarly journals Formin-based control of the actin cytoskeleton during cytokinesis

2013 ◽  
Vol 41 (6) ◽  
pp. 1750-1754 ◽  
Author(s):  
K. Adam Bohnert ◽  
Alaina H. Willet ◽  
David R. Kovar ◽  
Kathleen L. Gould
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Present Article ◽  
Actin Filaments ◽  
Terminal Event ◽  
Filamentous Actin ◽  
Daughter Cells ◽  
The Core ◽  
Diverse Species ◽  
Divided Genomes

Cytokinesis, the terminal event in the canonical cell cycle, physically separates daughter cells following mitosis. For cleavage to occur in many eukaryotes, a cytokinetic ring must assemble and constrict between divided genomes. Although dozens of different molecules localize to and participate within the cytokinetic ring, the core machinery comprises linear actin filaments. Accordingly, formins, which nucleate and elongate F-actin (filamentous actin) for the cytokinetic ring, are required for cytokinesis in diverse species. In the present article, we discuss specific modes of formin-based actin regulation during cell division and highlight emerging mechanisms and questions on this topic.

scholarly journals Anillin propels myosin-independent constriction of actin rings

2020 ◽  
Author(s):  
Ondřej Kučera ◽  
Daniel Janda ◽  
Valerie Siahaan ◽  
Sietske H. Dijkstra ◽  
Eliška Pilátová ◽  
...  
Keyword(s):  
Cell Division ◽  
Motor Activity ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Essential Step ◽  
Ring Constriction ◽  
Myosin Motors ◽  
Actin Rings

AbstractConstriction of the actin cytokinetic ring is an essential step of cell division. In a generally accepted view, the constriction is driven by relative sliding of actin filaments propelled by myosin motors. However, in multiple organisms, the ring constriction is myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that actin contractility can be propelled by anillin, a diffusible non-motor actin crosslinker, localising to the cytokinetic ring. We in vitro observed the formation and constriction of rings comprising multiple actin filaments bundled by anillin. Rings constricted due to anillin-generated forces maximising the overlap lengths between the filaments. Actin disassembly promoted constriction. We propose that actin crosslinkers, generating forces complementary to molecular motors, contribute to the contractility of diverse actin structures, including the cytokinetic ring.

scholarly journals Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae.

1996 ◽  
Vol 135 (6) ◽  
pp. 1535-1549 ◽  
Author(s):  
K L Hill ◽  
N L Catlett ◽  
L S Weisman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Critical Role ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuole Inheritance

During cell division, cytoplasmic organelles are not synthesized de novo, rather they are replicated and partitioned between daughter cells. Partitioning of the vacuole in the budding yeast Saccharomyces cerevisiae is coordinated with the cell cycle and involves a dramatic translocation of a portion of the parental organelle from the mother cell into the bud. While the molecular mechanisms that mediate this event are unknown, the vacuole's rapid and directed movements suggest cytoskeleton involvement. To identify cytoskeletal components that function in this process, vacuole inheritance was examined in a collection of actin mutants. Six strains were identified as being defective in vacuole inheritance. Tetrad analysis verified that the defect cosegregates with the mutant actin gene. One strain with a deletion in a myosin-binding region was analyzed further. The vacuole inheritance defect in this strain appears to result from the loss of a specific actin function; the actin cytoskeleton is intact and protein targeting to the vacuole is normal. Consistent with these findings, a mutation in the actin-binding domain of Myo2p, a class V unconventional myosin, abolishes vacuole inheritance. This suggests that Myo2p serves as a molecular motor for vacuole transport along actin filaments. The location of actin and Myo2p relative to the vacuole membrane is consistent with this model. Additional studies suggest that the actin filaments used for vacuole transport are dynamic, and that profilin plays a critical role in regulating their assembly. These results present the first demonstration that specific cytoskeletal proteins function in vacuole inheritance.

scholarly journals Spindle position dictates division site during asymmetric cell division in moss

2020 ◽  
Author(s):  
Elena Kozgunova ◽  
Mari W. Yoshida ◽  
Gohta Goshima
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Actin Filaments ◽  
Asymmetric Cell Division ◽  
Physcomitrella Patens ◽  
Plant Cells ◽  
Spindle Positioning ◽  
Division Site ◽  
Multicellular Organisms ◽  
Spindle Position

AbstractAsymmetric cell division (ACD) underlies the development of multicellular organisms. The division site in plant cells is predetermined prior to mitosis and the localization of the mitotic spindle is considered static, unlike in animal ACD, where the cell division site is defined by active spindle-positioning mechanisms. Here, we isolated a novel mutant of the microtubule-associated protein TPX2 in the moss Physcomitrella patens and observed abnormal spindle motility, which led to inverted asymmetric division during organ development. This phenotype was rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we identify an active spindle-positioning mechanism involving microtubules and actin filaments that sets the division site in plants, which is reminiscent of the acentrosomal ACD in animals, and suggests the existence of a common ancestral mechanism.

scholarly journals Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Shu-Zon Wu ◽  
Magdalena Bezanilla
Keyword(s):  
Cell Division ◽  
Motor Activity ◽  
Molecular Mechanism ◽  
Plant Cell ◽  
Actin Filaments ◽  
Plant Cells ◽  
Cell Plate ◽  
Cell Periphery ◽  
Division Site ◽  
Plant Cell Division

Plant cells divide using the phragmoplast, a microtubule-based structure that directs vesicles secretion to the nascent cell plate. The phragmoplast forms at the cell center and expands to reach a specified site at the cell periphery, tens or hundreds of microns distant. The mechanism responsible for guiding the phragmoplast remains largely unknown. Here, using both moss and tobacco, we show that myosin VIII associates with the ends of phragmoplast microtubules and together with actin plays a role in guiding phragmoplast expansion to the cortical division site. Our data lead to a model whereby myosin VIII links phragmoplast microtubules to the cortical division site via actin filaments. Myosin VIII's motor activity along actin provides a molecular mechanism for steering phragmoplast expansion.

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