Dynamics and organization of actin-labelled granules as a rapid transport mode of actin cytoskeleton components in Foraminifera

Abstract. Recent advances in fluorescent imaging facilitate actualistic studies on organisms used for palaeoceanographic reconstructions. Observations of cytoskeleton organization and dynamics in living foraminifera foster understanding of morphogenetic and biomineralization principles. This paper describes the organisation of a foraminiferal actin cytoskeleton using in vivo staining based on fluorescent SiR-actin. Surprisingly, the most distinctive feature in the organisation of actin in Foraminifera is the prevalence of actin-labelled granules (ALGs) within pseudopodial structures. Fluorescent signal obtained from granules dominate over dispersed signal from the actin meshwork. Actin-labelled granules are small (around 1 µm in diameter) actin-rich organelles demonstrating a wide range of motility behaviours from almost stationary oscillating around certain points to exhibiting rapid motion. These structures are present both in Globothalamea (Amphistegina, Ammonia) and Tubothalamea (Quinqueloculina). They are found to be active in all kinds of pseudopodial ectoplasmic structures, including granuloreticulopodia, globopodia, and lamellipodia, as well as within the endoplasm itself. Two hypotheses regarding their function are proposed: (1) They are involved in endocytosis and intracellular transport of different kinds of cargo; (2) They transport prefabricated and/or recycled actin fibres to the sites where they are needed. These hypothesis are not mutually exclusive. The first hypothesis is based on the presence of similar actin structures in fungi, fungi-like protists and some plant cells. The later hypothesis is based on the assumption that actin granules are analogous to tubulin paracrystals responsible for efficient transport of tubulin. Actin patches transported in that manner are most likely involved in maintaining shape, rapid reorganization, and elasticity of pseudopodial structures, as well as in adhesion to the substrate. Finally, our comparative studies suggest that a large proportion of actin-labelled granules probably represent fibrillar vesicles and elliptical fuzzy coated vesicles often identified in TEM images. Correlative fluorescent electron microscopic observations are proposed to verify this interpretation.

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SiR-actin-labelled granules in foraminifera: patterns, dynamics, and hypotheses

Biogeosciences ◽

10.5194/bg-17-995-2020 ◽

2020 ◽

Vol 17 (4) ◽

pp. 995-1011

Author(s):

Jan Goleń ◽

Jarosław Tyszka ◽

Ulf Bickmeyer ◽

Jelle Bijma

Keyword(s):

Electron Microscope ◽

Intracellular Transport ◽

Plant Cells ◽

Distinctive Pattern ◽

Transmission Electron ◽

Wide Range ◽

Microscope Images ◽

Set Up ◽

Tubulin Paracrystals

Abstract. Recent advances in fluorescence imaging facilitate actualistic studies of organisms used for palaeoceanographic reconstructions. Observations of cytoskeleton organisation and dynamics in living foraminifera foster understanding of morphogenetic and biomineralisation principles. This paper describes the organisation of a foraminiferal actin cytoskeleton using in vivo staining based on fluorescent SiR-actin. Surprisingly, the most distinctive pattern of SiR-actin staining in foraminifera is the prevalence of SiR-actin-labelled granules (ALGs) within pseudopodial structures. Fluorescent signals obtained from granules dominate over dispersed signals from the actin meshwork. SiR-actin-labelled granules are small (around 1 µm in diameter) actin-rich structures, demonstrating a wide range of motility behaviours, from almost stationarily oscillating around certain points to exhibiting rapid motion. These labelled microstructures are present both in Globothalamea (Amphistegina, Ammonia) and Tubothalamea (Quinqueloculina). They are found to be active in all kinds of pseudopodial ectoplasmic structures, including granuloreticulopodia, globopodia, and lamellipodia, as well as within the endoplasm. Several hypotheses are set up to explain either specific or non-specific actin staining. Two hypotheses regarding their function are proposed if specific actin labelling is taken into account: (1) granules are involved in endocytosis and intracellular transport of different kinds of cargo, or (2) they transport prefabricated and/or recycled actin fibres to the sites where they are needed. These hypotheses are not mutually exclusive. The first hypothesis is based on the presence of similar actin structures in fungi, fungi-like protists, and some plant cells. The later hypothesis is based on the assumption that actin granules are analogous to tubulin paracrystals responsible for efficient transport of tubulin. Actin patches transported in that manner are most likely involved in maintaining shape, rapid reorganisation, and elasticity of pseudopodial structures, as well as in adhesion to the substrate. Finally, our comparative studies suggest that a large proportion of SiR-actin-labelled granules probably represent fibrillar vesicles and elliptical fuzzy-coated vesicles often identified in transmission electron microscope images.

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Understanding Biological Structures Through Exploring the Mechanical Response of Cell-Like Systems

ASME 2008 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2008-192679 ◽

2008 ◽

Author(s):

Ying Zhang ◽

Philip R. LeDuc

Keyword(s):

Actin Cytoskeleton ◽

Actin Filaments ◽

Living Cell ◽

Cellular Response ◽

Cancer Metastasis ◽

Cell Structure ◽

Polymer Physics ◽

Wide Range

The actin cytoskeleton provides mechanical support for the cell and influences activities such as cancer metastasis and chemotaxis. While their mechanical responses have been studied in vivo and in vitro, understanding the link between these two forms remains challenging. To explore this gap and further understand cell structure, we reconstructed the cell cytoskeleton in a membrane-like spherical liposome to mimic the cellular environment; this enables an artificial “cell like” system. Through this approach, we are pursuing a path to compare in vitro mechanics from a polymer physics perspective of individual actin filaments with the in vivo mechanics of a living cell [1]. A living cell contains many organelles, which are in a highly packed environment and require significant organization to function. The actin cytoskeleton provides both structural and organizational regulation that is essential for cellular response. Here, we first encapsulated G-actin into giant unilamellar vesicles through an electroformation technique and then polymerized them into actin filaments (F-actin) within individual vesicles. To probe their conformation, we visualized these vesicles with fluorescence and laser scanning confocal microscopy. We then used a tapping mode atomic force microscopy to determine the mechanical properties of these cell-like systems. These results provide insight into a wide range of fields and studies including polymer physics, cell biology, and biotechnology.

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Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization

eLife ◽

10.7554/elife.49677 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Veronica Cloud ◽

Ada Thapa ◽

Pedro Morales-Sosa ◽

Tayla M Miller ◽

Sara A Miller ◽

...

Keyword(s):

Subcellular Localization ◽

Actin Cytoskeleton ◽

Spinocerebellar Ataxia Type ◽

Amino Terminus ◽

Heterozygous Mutation ◽

Polyglutamine Expansion ◽

Cytoskeleton Organization ◽

Protein Levels ◽

Spinocerebellar Ataxia Type 7

Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR.

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Regulation of Yeast Actin Cytoskeleton-Regulatory Complex Pan1p/Sla1p/End3p by Serine/Threonine Kinase Prk1p

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3759 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3759-3772 ◽

Cited By ~ 72

Author(s):

Guisheng Zeng ◽

Xianwen Yu ◽

Mingjie Cai

Keyword(s):

Actin Cytoskeleton ◽

Regulatory Protein ◽

Strong Support ◽

Stable Complex ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use ofPRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

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Gα12/13 regulate epiboly by inhibiting E-cadherin activity and modulating the actin cytoskeleton

The Journal of Cell Biology ◽

10.1083/jcb.200805148 ◽

2009 ◽

Vol 184 (6) ◽

pp. 909-921 ◽

Cited By ~ 48

Author(s):

Fang Lin ◽

Songhai Chen ◽

Diane S. Sepich ◽

Jennifer Ray Panizzi ◽

Sherry G. Clendenon ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Molecular Mechanisms ◽

Genetic Studies ◽

Yolk Cell ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization ◽

Cell Layers ◽

Dependent Pathway ◽

E Cadherin

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Gα12/13 signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Gα12/13 regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Gα12/13 modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Gα12/13 regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

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Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

Biochemical Society Transactions ◽

10.1042/bst0380823 ◽

2010 ◽

Vol 38 (3) ◽

pp. 823-828 ◽

Cited By ~ 8

Author(s):

Tijs Ketelaar ◽

Hannie S. van der Honing ◽

Anne Mie C. Emons

Keyword(s):

Actin Cytoskeleton ◽

Physical Properties ◽

Optical Tweezers ◽

Intracellular Transport ◽

Plant Cells ◽

Actin Dynamics ◽

Actin Binding ◽

Structural Basis ◽

Actin Organization ◽

Cytoplasmic Organization

In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be considered. In the present review, we discuss literature that gives an insight into how cytoplasmic organization is achieved and in which actin-binding proteins have been identified that play a role in this process. We discuss how physical properties of the actin cytoskeleton in the cytoplasm of live plant cells, such as deformability and elasticity, can be probed by using optical tweezers. This technique allows non-invasive manipulation of cytoplasmic organization. Optical tweezers, integrated in a confocal microscope, can be used to manipulate cytoplasmic organization while studying actin dynamics. By combining this with mutant studies and drug applications, insight can be obtained about how the physical properties of the actin cytoskeleton, and thus the cytoplasmic organization, are influenced by different cellular processes.

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Actin modulates shape and mechanics of tubular membranes

Science Advances ◽

10.1126/sciadv.aaz3050 ◽

2020 ◽

Vol 6 (17) ◽

pp. eaaz3050

Author(s):

A. Allard ◽

M. Bouzid ◽

T. Betz ◽

C. Simon ◽

M. Abou-Ghali ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Mammalian Cells ◽

Intracellular Transport ◽

Plant Cells ◽

Tube Surface ◽

Apparent Contradiction ◽

Single Tube ◽

Tubular Membrane ◽

Tubular Membranes

The actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes for intracellular transport of material in mammalian cells, yeast, or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are formed during this process and destabilize into vesicles. While the role of actin in tubule destabilization through scission is suggested, literature also provides examples of actin-mediated stabilization of membranous structures. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatiotemporally. Depending on network cohesiveness, actin is able to entirely stabilize or locally maintain membrane tubes under pulling. On a single tube, thicker portions correlate with the presence of actin. These structures relax over several minutes and may provide enough time and curvature geometries for other proteins to act on tube stability.

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Actin modulates shape and mechanics of tubular membranes

10.1101/712505 ◽

2019 ◽

Author(s):

A. Allard ◽

M. Bouzid ◽

T. Betz ◽

C. Simon ◽

M. Abou-Ghali ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Mammalian Cells ◽

Intracellular Transport ◽

Plant Cells ◽

Tube Surface ◽

Apparent Contradiction ◽

Single Tube ◽

Tubular Membrane ◽

Tubular Membranes

The actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes in intracellular transport of material in mammalian cells, yeast or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are involved during these processes, and destabilize into vesicles. While the role of actin in this destabilization process is still debated, literature also provide examples of membranous structures stabilization by actin. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatio-temporally. Depending on network cohesiveness, actin is able to stabilize, or maintain membrane tubes under pulling. Indeed, on a single tube, thicker portions correlate with the presence of actin. Such structures relax over several minutes, and may provide enough time and curvature geometries for other proteins to act on tube stability.

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Sac1 Lipid Phosphatase and Stt4 Phosphatidylinositol 4-Kinase Regulate a Pool of Phosphatidylinositol 4-Phosphate That Functions in the Control of the Actin Cytoskeleton and Vacuole Morphology

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2396 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2396-2411 ◽

Cited By ~ 161

Author(s):

Michelangelo Foti ◽

Anjon Audhya ◽

Scott D. Emr

Keyword(s):

Actin Cytoskeleton ◽

Temperature Sensitive ◽

Primary Role ◽

Cytoskeleton Organization ◽

Phosphoinositide Phosphatase ◽

Actin Cytoskeleton Organization ◽

Sensitive Allele ◽

Phosphatidylinositol 4 ◽

Mutant Cells

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1tsallele and one of two temperature-sensitive PtdIns 4-kinase genes,stt4tsor pik1ts, we have demonstrated that the bulk of PtdIns(4)P that accumulates insac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated withstt4tsbut notpik1tsmutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1tsmutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.

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The Role of the Tethering Proteins p115 and GM130 in Transport through the Golgi Apparatus In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.635 ◽

2000 ◽

Vol 11 (2) ◽

pp. 635-645 ◽

Cited By ~ 134

Author(s):

Joachim Seemann ◽

Eija Jämsä Jokitalo ◽

Graham Warren

Keyword(s):

Intracellular Transport ◽

Microscopic Analysis ◽

Electron Microscopic ◽

Golgi Membranes ◽

Transport Vesicles ◽

Golgi Region ◽

Quantitative Immunofluorescence ◽

Target Membrane ◽

Mitotic Phosphorylation

Biochemical data have shown that COPI-coated vesicles are tethered to Golgi membranes by a complex of at least three proteins: p115, giantin, and GM130. p115 binds to giantin on the vesicles and to GM130 on the membrane. We now examine the function of this tethering complex in vivo. Microinjection of an N-terminal peptide of GM130 or overexpression of GM130 lacking this N-terminal peptide inhibits the binding of p115 to Golgi membranes. Electron microscopic analysis of single microinjected cells shows that the number of COP-sized transport vesicles in the Golgi region increases substantially, suggesting that transport vesicles continue to bud but are less able to fuse. This was corroborated by quantitative immunofluorescence analysis, which showed that the intracellular transport of the VSV-G protein was significantly inhibited. Together, these data suggest that this tethering complex increases the efficiency with which transport vesicles fuse with their target membrane. They also provide support for a model of mitotic Golgi fragmentation in which the tethering complex is disrupted by mitotic phosphorylation of GM130.

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