scholarly journals Cucurbitacins: elucidation of their interactions with the cytoskeleton

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3357 ◽  
Author(s):  
Xiaojuan Wang ◽  
Mine Tanaka ◽  
Herbenya Silva Peixoto ◽  
Michael Wink
Keyword(s):  
Actin Filaments ◽  
Living Cells ◽  
Cytoskeletal Proteins ◽  
Microtubule Assembly ◽  
Tubulin Polymerization ◽  
Mitotic Spindles ◽  
Cucurbitacin B ◽  
M Phase ◽  
Cucurbitacin E

Cucurbitacins, a class of toxic tetracyclic triterpenoids in Cucurbitaceae, modulate many molecular targets. Here we investigated the interactions of cucurbitacin B, E and I with cytoskeletal proteins such as microtubule and actin filaments. The effects of cucurbitacin B, E and I on microtubules and actin filaments were studied in living cells (Hela and U2OS) and in vitro using GFP markers, immunofluorescence staining and in vitro tubulin polymerization assay. Cucurbitacin B, E and I apparently affected microtubule structures in living cells and cucurbitacin E inhibited tubulin polymerization in vitro with IC50 value of 566.91 ± 113.5 µM. Cucurbitacin E did not affect the nucleation but inhibited the growth phase and steady state during microtubule assembly in vitro. In addition, cucurbitacin B, E and I all altered mitotic spindles and induced the cell cycle arrest at G2/M phase. Moreover, they all showed potent effects on actin cytoskeleton by affecting actin filaments through the depolymerization and aggregation. The interactions of cucubitacin B, E and I with microtubules and actin filaments present new insights into their modes of action.

Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

1995 ◽  
Vol 53 ◽  
pp. 902-903
Author(s):  
J. R. Kuhn ◽  
M. Poenie
Keyword(s):  
Mitotic Spindle ◽  
Actin Filaments ◽  
Cell Shape ◽  
Dynamic Structure ◽  
Living Cells ◽  
Cytoskeletal Proteins ◽  
Polarization Microscopy ◽  
Video Enhancement ◽  
Ultrastructural Studies ◽  
Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

scholarly journals Dynamic Localization of CLIP-170 to Microtubule Plus Ends Is Coupled to Microtubule Assembly

1999 ◽  
Vol 144 (1) ◽  
pp. 99-112 ◽  
Author(s):  
Georgios S. Diamantopoulos ◽  
Franck Perez ◽  
Holly V. Goodson ◽  
Gérard Batelier ◽  
Ronald Melki ◽  
...  
Keyword(s):  
Sedimentation Velocity ◽  
Microtubule Assembly ◽  
In Vitro Assays ◽  
Cross Linking ◽  
Tubulin Polymerization ◽  
Binding Properties ◽  
Dynamic Localization ◽  
Microtubule Binding

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.

scholarly journals CBMT-12. FOCAD LOSS IMPACTS MICROTUBULE ASSEMBLY, G2/M PROGRESSION AND PATIENT SURVIVAL IN ASTROCYTIC GLIOMAS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi35-vi35
Author(s):  
Frank Brand ◽  
Alisa Förster ◽  
Anne Kosfeld ◽  
Martin Bucher ◽  
Carina Thomé ◽  
...  
Keyword(s):  
Overall Survival ◽  
Molecular Mechanisms ◽  
Methylation Status ◽  
Malignant Gliomas ◽  
Microtubule Assembly ◽  
Copy Number Loss ◽  
Isocitrate Dehydrogenase 1 ◽  
M Phase ◽  
Astrocytic Gliomas

Abstract In search of novel genes associated with glioma pathogenesis, we have previously shown that KIAA1797/FOCAD is frequently deleted in malignant gliomas and that the encoded focadhesin functions as a tumor suppressor impacting proliferation and migration of glioma cells in vitro and in vivo. Here, we examine an association of FOCAD copy number loss with overall survival of patients with astrocytic gliomas, and address the molecular mechanisms that govern the suppressive effect of focadhesin on glioma growth. FOCAD loss was associated with inferior outcome in patients with isocitrate dehydrogenase 1 or 2 (IDH)-mutant astrocytic gliomas of WHO grades II-IV. Multivariate analysis considering age at diagnosis, IDH mutation and MGMT promoter methylation status confirmed FOCAD loss as a prognostic factor for overall survival. Using a yeast two-hybrid screen and pull-down assays, we identified tubulin beta-6 and other tubulin family members as novel focadhesin-interacting partners. We demonstrate that tubulins and focadhesin co-localize at the centrosome and that focadhesin is enriched in proximity to centrioles. Focadhesin is recruited to microtubules via its interaction partner SLAIN motif family member 2 and reduces microtubule assembly rates, possibly explaining why focadhesin decreases cell migration. During the cell cycle, focadhesin levels peak in G2/M phase and influence time-dependent G2/M progression possibly via polo like kinase 1 phosphorylation, providing an explanation for focadhesin-dependent cell growth reduction. We conclude that FOCAD loss may promote biological aggressiveness and worsen clinical outcome of diffuse astrocytic gliomas by enhancing microtubule assembly and accelerating G2/M phase progression.

scholarly journals Arl2 and Arl3 Regulate Different Microtubule-dependent Processes

2006 ◽  
Vol 17 (5) ◽  
pp. 2476-2487 ◽  
Author(s):  
Chengjing Zhou ◽  
Leslie Cunningham ◽  
Adam I. Marcus ◽  
Yawei Li ◽  
Richard A. Kahn
Keyword(s):  
Common Ancestor ◽  
Tubulin Polymerization ◽  
Mitotic Spindles ◽  
Eukaryotic Evolution ◽  
Cycle Arrest ◽  
Initial Characterization ◽  
M Phase ◽  
Functional Features ◽  
Dependent Processes

Arl2 and Arl3 are closely related members of the Arf family of regulatory GTPases that arose from a common ancestor early in eukaryotic evolution yet retain extensive structural, biochemical, and functional features. The presence of Arl3 in centrosomes, mitotic spindles, midzones, midbodies, and cilia are all supportive of roles in microtubule-dependent processes. Knockdown of Arl3 by siRNA resulted in changes in cell morphology, increased acetylation of α-tubulin, failure of cytokinesis, and increased number of binucleated cells. We conclude that Arl3 binds microtubules in a regulated manner to alter specific aspects of cytokinesis. In contrast, an excess of Arl2 activity, achieved by expression of the [Q70L]Arl2 mutant, caused the loss of microtubules and cell cycle arrest in M phase. Initial characterization of the underlying defects suggests a defect in the ability to polymerize tubulin in the presence of excess Arl2 activity. We also show that Arl2 is present in centrosomes and propose that its action in regulating tubulin polymerization is mediated at centrosomes. Somewhat paradoxically, no phenotypes were observed Arl2 expression was knocked down or Arl3 activity was increased in HeLa cells. We conclude that Arl2 and Arl3 have related but distinct roles at centrosomes and in regulating microtubule-dependent processes.

The effect of cytoskeletal protein mutations on cell motility and morphogenesis

1992 ◽  
Vol 50 (1) ◽  
pp. 594-595
Author(s):  
David A. Knecht
Keyword(s):  
Cell Motility ◽  
Actin Filaments ◽  
Cytoskeletal Proteins ◽  
Cross Linking ◽  
Cytoskeletal Protein ◽  
Homologous Gene ◽  
Complex Interactions ◽  
Associated Proteins ◽  
Protein Mutations

The cortical cytoskeleton of eukaryotic cells is composed of actin filaments and a variety of associated proteins. The polymerization, depolymerization, cross-linking and bundling of these filaments, are presumed to be intimately involved in such processes as cell motility, cell adhesion and cell shape. In developing systems, all of these processes are involved in the morphogenetic mechanisms that shape tissues, organs and organisms.We are investigating the complex interactions among cytoskeletal proteins using the simple eukaryotic amoebae, Dictyostelium discoideum. Our approach is to determine the function of the components of the cytoskeleton by creating mutants lacking particular proteins, or containing specific alterations in these proteins. Mutants lacking myosin heavy chain have been created using antisense RNA and homologous gene targetting. These cells have alterations in their shape and movement, and are incapable of accomplishing normal morphogenesis. Another cytoskeletal protein is ABP-120, which is capable of cross-linking actin filaments into orthogonal arrays, leading to the formation of an actin gel in vitro. ABP-120 is found in newly formed pseudopods extended during the chemotactic respose to extracellular cAMP. Mutants lacking this protein have been created by disruption of the chromosomal gene with a transformation vector. These cells are not as dramatically affected as the myosin mutants, but have clear alterations in their motility and in the pathway of responses in the cytoskeleton that correlate with the expected function of this protein. Mutations in several other cytoskeletal genes are currently being constructed.

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

1993 ◽  
Vol 105 (3) ◽  
pp. 765-775 ◽  
Author(s):  
E. Friederich ◽  
T.E. Kreis ◽  
D. Louvard
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Site ◽  
Actin Filaments ◽  
Living Cells ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Fast Growing ◽  
Actin Binding Site

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

scholarly journals Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis

2005 ◽  
Vol 170 (7) ◽  
pp. 1057-1066 ◽  
Author(s):  
Isabel Peset ◽  
Jeanette Seiler ◽  
Teresa Sardon ◽  
Luis A. Bejarano ◽  
Sonja Rybina ◽  
...  
Keyword(s):  
Translational Control ◽  
Microtubule Assembly ◽  
Aurora A Kinase ◽  
Microtubule Stabilization ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
M Phase ◽  
Microtubule Growth ◽  
And Function

The Xenopus protein Maskin has been previously identified and characterized in the context of its role in translational control during oocyte maturation. Maskin belongs to the TACC protein family. In other systems, members of this family have been shown to localize to centrosomes during mitosis and play a role in microtubule stabilization. Here we have examined the putative role of Maskin in spindle assembly and centrosome aster formation in the Xenopus egg extract system. Depletion and reconstitution experiments indicate that Maskin plays an essential role for microtubule assembly during M-phase. We show that Maskin interacts with XMAP215 and Eg2, the Xenopus Aurora A kinase in vitro and in the egg extract. We propose that Maskin and XMAP215 cooperate to oppose the destabilizing activity of XKCM1 therefore promoting microtubule growth from the centrosome and contributing to the determination of microtubule steady-state length. Further more, we show that Maskin localization and function is regulated by Eg2 phosphorylation.

scholarly journals Tryprostatin A, a specific and novel inhibitor of microtubule assembly

1998 ◽  
Vol 333 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Takeo USUI ◽  
Masuo KONDOH ◽  
Cheng-Bin CUI ◽  
Tadanori MAYUMI ◽  
Hiroyuki OSADA
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Progression ◽  
Microtubule Assembly ◽  
Inhibition Mechanism ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Terminal Domain ◽  
M Phase

We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 µM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 µM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.

scholarly journals Specific association of an M-phase kinase with isolated mitotic spindles and identification of two of its substrates as MAP4 and MAP1B.

1991 ◽  
Vol 2 (11) ◽  
pp. 861-874 ◽  
Author(s):  
R M Tombes ◽  
J G Peloquin ◽  
G G Borisy
Keyword(s):  
Mitotic Cycle ◽  
Chinese Hamster ◽  
Mitotic Spindles ◽  
Microtubule Associated Proteins ◽  
M Phase ◽  
Associated Proteins ◽  
Specific Association ◽  
Cycle Dependent

Isolated mammalian (Chinese hamster ovary [CHO]) metaphase spindles were found to be enriched in a histone H1 kinase whose activity was mitotic-cycle dependent. Two substrates for the kinase were identified as MAP1B and MAP4. Partially purified spindle kinase retained activity for the spindle microtubule-associated proteins (MAPs) as well as brain and other tissue culture MAPs; on phosphorylation, spindle MAPs exhibited increased immunoreactivity with MPM-2, a monoclonal antibody specific for a subset of mitotic phosphoproteins. Immunofluorescence using an anti-thiophosphoprotein antibody localized in vitro phosphorylated spindle proteins to microtubule fibers, centrosomes, kinetochores, and midbodies. The fractionated spindle kinase was reactive with anti-human p34cdc2 antibodies and with an anti-human cyclin B but not an anti-human cyclin A antibody. We conclude that spindle MAPs undergo mitotic cycle-dependent phosphorylations in vivo and associate with a kinase that remains active on spindle isolation and may be related to p34cdc2.

scholarly journals XMAP230 is required for normal spindle assembly in vivo and in vitro

1999 ◽  
Vol 112 (23) ◽  
pp. 4337-4346 ◽  
Author(s):  
B. Cha ◽  
L. Cassimeris ◽  
D.L. Gard
Keyword(s):  
Spindle Assembly ◽  
Chromosome Loss ◽  
Mitotic Spindles ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
M Phase ◽  
Spindle Microtubules ◽  
Bipolar Spindles

XMAP230 is a high molecular mass microtubule-associated protein isolated from Xenopus oocytes and eggs, and has been recently shown to be a homolog of mammalian MAP4. Confocal immunofluorescence microscopy revealed that XMAP230 is associated with microtubules throughout the cell cycle of early Xenopus embryos. During interphase XMAP230 is associated with the radial arrays of microtubules and midbodies remaining from the previous division. During mitosis, XMAP230 is associated with both astral microtubules and microtubules of the central spindle. Microinjection of affinity-purified anti-XMAP230 antibody into blastomeres severely disrupted the assembly of mitotic spindles during the rapid cleavage cycles of early development. Both monopolar half spindles and bipolar spindles were assembled from XMAP230-depleted extracts in vitro. However, spindles assembled in XMAP230-depleted extracts exhibited a reduction in spindle width, reduced microtubule density, chromosome loss, and reduced acetylation of spindle MTs. Similar defects were observed in the spindles assembled in XMAP230-depleted extracts that had been cycled through interphase. Depletion of XMAP230 had no effect on the pole-to-pole length of spindles, and depletion of XMAP230 from both interphase and M-phase extracts had no effect on the rate of microtubule elongation. From these results, we conclude that XMAP230 plays an important role in normal spindle assembly, primarily by acting to stabilize spindle microtubules, and that the observed defects in spindle assembly may result from enhanced microtubule dynamics in XMAP230-depleted extracts.

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