Localization of myosin Va is dependent on the cytoskeletal organization in the cell

2001 ◽  
Vol 79 (1) ◽  
pp. 93-106 ◽  
Author(s):  
Corinne Lionne ◽  
Folma Buss ◽  
Tony Hodge ◽  
Gudrun Ihrke ◽  
John Kendrick-Jones
Keyword(s):  
Membrane Trafficking ◽  
Molecular Motors ◽  
Leading Edge ◽  
Apical Surface ◽  
Myosin V ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Myosin Va ◽  
Dynamic Membranes ◽  
In Cells

Myosin V plays an important role in membrane trafficking events. Its implication in the transport of pigment granules in melanocytes and synaptic vesicles in neurons is now well established. However, less is known about its function(s) in other cell types. Finding a common function is complicated by the diversity of myosin V expression in different tissues and organisms and by its association with different subcellular compartments. Here we show that myosin V is present in a variety of cells. Within the same cell type under different physiological conditions, we observed two main cellular locations for myosin V that were dependent on the dynamics of the plasma membrane: in cells with highly dynamic membranes, myosin V was specifically concentrated at the leading edge in membrane ruffles, whereas in cells with less dynamic membranes, myosin V was enriched around the microtubule-organizing center. The presence of myosin V in the leading ruffling edge of the cell was induced by growth factor stimulation and was dependent on the presence of a functional motor domain. Moreover, myosin V localization at the microtubule-organizing center was dependent on the integrity of the microtubules. In polarized epithelial cells (WIF-B), where the microtubule-organizing region is close to the actin-rich apical surface, one single pool of myosin V, sensitive to the integrity of both microtubules and actin filaments, was observed.Key words: cell motility, cytoskeleton dynamics, molecular motors, mouse brain unconventional myosin Va, ruffles.

scholarly journals Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

1979 ◽  
Vol 83 (3) ◽  
pp. 623-632 ◽  
Author(s):  
M Schliwa ◽  
U Euteneuer ◽  
W Herzog ◽  
K Weber
Keyword(s):  
Complete Removal ◽  
Cell System ◽  
The State ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Functional Changes ◽  
Organizing Center ◽  
Pigment Distribution ◽  
And Function ◽  
In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A

1993 ◽  
Vol 106 (3) ◽  
pp. 789-802 ◽  
Author(s):  
M. Roa ◽  
V. Cornet ◽  
C.Z. Yang ◽  
B. Goud
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Atp Depletion ◽  
Cell Fractionation ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Organizing Center ◽  
Golgi Network

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10–15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

Subcellular localization of GFP-myosin-V in live mouse melanocytes

1999 ◽  
Vol 112 (17) ◽  
pp. 2853-2865 ◽  
Author(s):  
V. Tsakraklides ◽  
K. Krogh ◽  
L. Wang ◽  
J.C. Bizario ◽  
R.E. Larson ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Actin Filaments ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Motor Domain ◽  
Chick Brain ◽  
Myosin V ◽  
Myosin Va ◽  
Myosin Vb ◽  
In Cells

Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.

scholarly journals Tubular early endosomal networks in AtT20 and other cells.

1991 ◽  
Vol 115 (3) ◽  
pp. 635-653 ◽  
Author(s):  
J Tooze ◽  
M Hollinshead
Keyword(s):  
Electron Microscope ◽  
Fluid Phase ◽  
Microtubule Organizing Center ◽  
Local Networks ◽  
Late Endosomes ◽  
Interphase Cells ◽  
Organizing Center ◽  
Mdck I ◽  
Att20 Cell ◽  
In Cells

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.

scholarly journals Associations of elements of the Golgi apparatus with microtubules.

1984 ◽  
Vol 99 (3) ◽  
pp. 1092-1100 ◽  
Author(s):  
A A Rogalski ◽  
S J Singer
Keyword(s):  
Golgi Apparatus ◽  
Cultured Cells ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Free Microtubules ◽  
Vesicular Stomatitis ◽  
In Cells

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.

scholarly journals Growth Cone-Localized Microtubule Organizing Center Establishes Microtubule Orientation in Dendrites

2019 ◽  
Author(s):  
Xing Liang ◽  
Marcela Kokes ◽  
Richard Fetter ◽  
Melissa A. Pickett ◽  
Maria D. Sallee ◽  
...  
Keyword(s):  
Growth Cone ◽  
Membrane Trafficking ◽  
Dendritic Growth ◽  
Microtubule Organizing Center ◽  
Microtubule Nucleation ◽  
Recycling Endosomes ◽  
Microtubule Orientation ◽  
Microtubule Polarity ◽  
Organizing Center ◽  
Subcellular Compartmentalization

AbstractA polarized arrangement of neuronal microtubule arrays is the foundation of membrane trafficking and subcellular compartmentalization. Conserved among both invertebrates and vertebrates, axons contain exclusively “plus-end-out” microtubules while dendrites contain a high percentage of “minus-end-out” microtubules, the origins of which have been a mystery. Here we show that the dendritic growth cone contains a non-centrosomal microtubule organizing center (ncMTOC), which generates minus-end-out microtubules along outgrowing dendrites and plus-end-out microtubules in the growth cone. RAB-11-positive recycling endosomes accumulate in this region and are responsible for localizing the microtubule nucleation complex γ-TuRC. The MTOC tracks the extending growth cone by kinesin-1/UNC-116-mediated endosome movements on distal plus-end-out microtubules and dynein-mediated endosome clustering near MTOC. Critically, perturbation of the function or localization of the MTOC causes reversed microtubule polarity in dendrites. These findings unveil the dendritic MTOC as a critical organelle for establishing axon-dendrite polarity.

scholarly journals Growth cone-localized microtubule organizing center establishes microtubule orientation in dendrites

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xing Liang ◽  
Marcela Kokes ◽  
Richard D Fetter ◽  
Maria Danielle Sallee ◽  
Adrian W Moore ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Growth Cone ◽  
Membrane Trafficking ◽  
Dendritic Growth ◽  
Microtubule Organizing Center ◽  
Microtubule Nucleation ◽  
Microtubule Orientation ◽  
Microtubule Polarity ◽  
Organizing Center ◽  
Subcellular Compartmentalization

A polarized arrangement of neuronal microtubule arrays is the foundation of membrane trafficking and subcellular compartmentalization. Conserved among both invertebrates and vertebrates, axons contain exclusively ‘plus-end-out’ microtubules while dendrites contain a high percentage of ‘minus-end-out’ microtubules, the origins of which have been a mystery. Here we show that in Caenorhabditis elegans the dendritic growth cone contains a non-centrosomal microtubule organizing center (MTOC), which generates minus-end-out microtubules along outgrowing dendrites and plus-end-out microtubules in the growth cone. RAB-11-positive endosomes accumulate in this region and co-migrate with the microtubule nucleation complex γ-TuRC. The MTOC tracks the extending growth cone by kinesin-1/UNC-116-mediated endosome movements on distal plus-end-out microtubules and dynein clusters this advancing MTOC. Critically, perturbation of the function or localization of the MTOC causes reversed microtubule polarity in dendrites. These findings unveil the endosome-localized dendritic MTOC as a critical organelle for establishing axon-dendrite polarity.

scholarly journals Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

2009 ◽  
Vol 20 (22) ◽  
pp. 4816-4825 ◽  
Author(s):  
Stefan Koch ◽  
Christopher T. Capaldo ◽  
Stanislav Samarin ◽  
Porfirio Nava ◽  
Irmgard Neumaier ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Leading Edge ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Interfering Rna

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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