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PubMed1993

Actin and cell movement.

The primary locomotory organelle of non-muscle, eukaryotic cells is the lamellipodium, a thin layer of cytoplasm that exhibits active protrusive activity. Earlier studies have implicated actin polymerization in the formation of lamellipodia, whereby actin monomers insert at the front and dissociate at the rear, in a treadmilling fashion. However, other models based on gel swelling and a breakdown of actin networks at the site of protrusion of the lamellipodium have also been proposed. By employing videomicroscopy and electron microscopy of the same cells, in this case mouse macrophages, it could be shown that lamellipodial protrusion is directly linked with the growth of dense actin meshworks. The gel swelling and cortical breakdown models are not supported by this data. Using rapidly locomoting fish keratocytes, Theriot and Mitchison (Nature 352, 126-131, 1991) recently obtained results that they interpreted as supporting a form of actin filament dynamics different from treadmilling. In their new "nucleation release model" the actin filaments in the lamellipodium are predicted as being very short (less than 0.5 micron) and randomly organized. We have now investigated the ultrastructure of the keratocyte cytoskeleton. Our results show that the actin filaments in these cells are very long and organized in dense and regular, more or less orthogonal networks. A gradient of filament density across the rear part of the lamellipodium suggests that the filaments are graded in length. These data support a treadmilling type model for the eratocyte.

Actins↗
PubMed1990

Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement.

The regulation of the internal open reading frame (ORF) of tobacco mosaic virus (TMV) that encodes the 30K movement protein was examined by constructing mutants in vitro with the putative coat protein subgenomic RNA promoter and leader sequences inserted upstream of the 30K ORF. A mutant with a 49-nucleotide fragment of the promoter region inserted replicated only transiently before being overtaken by a progeny wild-type virus with the insert deleted. A mutant with a 253-nucleotide promoter region fragment inserted replicated stably, and the inserted promoter was active in its new location. The production of 30K protein was not enhanced by this promoter/leader insertion to a level similar to that of coat protein. However, the accumulation of 30K protein was delayed, suggesting that different promoters/leader sequences determine the time of expression of the genes. This mutant was deficient in movement. A similar mutant, but with increased production of 30K protein, overcame the movement deficiency, suggesting that 30K protein is needed during the early stages of infection for efficient cell-to-cell movement of the virus.

Base Sequence↗
PubMed2004

The role of Rho GTPases in the regulation of the rearrangement of actin cytoskeleton and cell movement.

The rearrangement of the actin cytoskeleton has been shown to play a critical role in the development of transformation and malignant phenotype of cancer cells. Rho family GTPases regulate the arrangement of the actin cytoskeleton. By wound-healing assay, we have found that NIH 3T3 fibroblast cells move towards the wound- gaps by extending filopodial and lamellipdial structures at the leading edge of the moving cells. We have inactivated the function of Rho GTPases of v-Ras transformed NIH 3T3 cells by overexpressing Rho GTPase-activating (RhoGAP) domain of RhoGAP of p190. We have observed that inactivation of Rho, Rac and Cdc42 GTPases by overexpressing RHG causes inhibition of: (i) polymerization of actin to form filaments, (ii) formation of lamellipodia, filopodia and stress fibres, (iii) cell motility, (iv) cell spreading and (v) cell-to-cell adhesions. These results further strengthen the current knowledge on the role of Rho, Rac and Cdc42 GTPases in the regulation of the rearrangement of actin cytoskeleton. Our results, for the first time, demonstrate that RhoGAP domain of RhoGAP could be used to study the molecular mechanism of Ras-mediated signalling in growth, differentiation and carcinogenesis.

Actin Cytoskeleton↗
PubMed2006

Analysis and visualization of cell movement in the developing zebrafish brain.

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

Animals↗
PubMed1984

Saccade-related neurons in cat superior colliculus: pandirectional movement cells with postsaccadic responses.

The superior colliculus is known to contain cells discharging before saccadic eye movements as well as cells responding to sensory stimuli. In this study extracellular single unit recordings were made in the alert trained cat with the head fixed. A novel type of eye movement-related response was found in 9% (32/344) of the cells recorded. These cells differ from previously reported eye movement-related neurons in the timing of their discharge, which accompanies but does not precede saccades. The timing of discharge varies across units from less than 10 ms after the onset of eye movement to as much as 80 ms. Comparable latencies were found regardless of whether saccades were directed contralateral or ipsilateral to the recording site. Most units have an abrupt onset of discharge, but some show a very gradual increase in discharge rate. Most cells (69% or 22/32) discharged with equal vigor for all saccades, regardless of direction. The remainder tended to show higher-frequency bursts when saccades were directed contralaterally, but even these units were not encoding saccade direction by their pattern of discharge. Thus the discharge pattern could be summarized as an omnidirectional burst. For the vast majority of cells (81% or 26/32) the duration of discharge did not correlate with the duration of eye movement. The same pattern of firing was seen with saccades in light and in complete darkness. Thus the saccade-related discharge was not due to changes in visual stimulation during saccades. A minority of the units (15% or 5 of 32) that discharged with but not before saccades also responded to visual stimuli in the absence of eye movements. Saccade-related activity was dependent on alertness of the cat, as measured by behavioral performance and EEG. The close temporal association between saccades and unit discharge disappeared during drowsiness. These units could be reflecting either proprioceptive or corollary discharge signals to the superior colliculus. However, their response properties differ somewhat from those found in previous studies of proprioceptive inputs to the colliculus. Such differences could be due to the effects of the anesthetics that were used in studies of proprioceptive responses. Alternatively, the cells reported here could be conveying a corollary discharge signal. These cells occurred in patches or clusters. This is consistent with a wealth of anatomic data indicating a modular organization of the colliculus.

Animals↗
PubMed2003

Chemotactic cell movement during development.

Chemotaxis is an important mechanism controlling cell migration over either short or long distances during different developmental processes. Small rapid diffusing chemo-attractants are detected through serpentine, G protein coupled receptors through graded activation of receptors along the length of the cell. Internal amplification results in polarisation of the actin and myosin cytoskeletal dynamics along the gradient and directed movement. The dynamics of these processes can now be studied in individual cells in developing organisms. Slow diffusing chemo-attractants such as growth factors, providing short-range guidance information, often signal through tyrosine kinase receptors. Detection of these signals may involve the active extension of very long cellular process up growth factor gradients, followed by translocation of the cell in the direction of the gradient.

Animals↗
PubMed2001

Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus.

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3), and betad (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

Cell Membrane↗
PubMed2001

Cucumber mosaic virus-plant interactions: identification of 3a protein sequences affecting infectivity, cell-to-cell movement, and long-distance movement.

Mutants of the Cucumber mosaic virus (CMV) movement protein (MP) were generated and analyzed for their effects on virus movement and pathogenicity in vivo. Similar to the wild-type MP, mutants M1, M2, and M3, promoted virus movement in eight plant species. Mutant M3 showed some differences in pathogenicity in one host species. Mutant M8 showed some host-specific alterations in movement in two hypersensitive hosts of CMV. Mutant M9 showed altered pathogenicity on three hosts and was temperature sensitive for long-distance movement, demonstrating that cell-to-cell and long-distance movement are distinct movement functions for CMV. Four mutants (M4, M5, M6, and M7) were debilitated from movement in all hosts tested. Mutants M4, M5, and M6 could be complemented in trans by the wild-type MP expressed transgenically, although not by each other or by mutant M9 (at the restrictive temperature). Mutant M7 showed an inability to be complemented in trans. From these mutants, different aspects of the CMV movement process could be defined and specific roles for particular sequence domains assigned. The broader implications of these functions are discussed.

Alanine↗
PubMed1995

Characterisation of cell movement by impedance measurement on fibroblasts grown on perforated Si-membranes.

Mouse fibroblasts grown on perforated Si-membranes (pore diameter approximately 10 microns have been studied to clarify cell locomotive ability. The cell motility was microscopically monitored by a time-lapse video system and, simultaneously, the impedance of the growing cells was measured every 5 s. The correlations between observed cell activities and measured impedance events are discussed and classified. The method is sensitive and allows discrimination between signals arising from translocation of single cells and those arising from filopodia activities. Both cell and filopodia motion could be detected. Designs of microdevices fabricated in semiconductor technology are presented.

3T3 Cells↗
PubMed2004

Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis.

Spermatogenesis is the process by which a single spermatogonium develops into 256 spermatozoa, one of which will fertilize the ovum. Since the 1950s when the stages of the epithelial cycle were first described, reproductive biologists have been in pursuit of one question: How can a spermatogonium traverse the epithelium, while at the same time differentiating into elongate spermatids that remain attached to the Sertoli cell throughout their development? Although it was generally agreed upon that junction restructuring was involved, at that time the types of junctions present in the testis were not even discerned. Today, it is known that tight, anchoring, and gap junctions are found in the testis. The testis also has two unique anchoring junction types, the ectoplasmic specialization and tubulobulbar complex. However, attention has recently shifted on identifying the regulatory molecules that "open" and "close" junctions, because this information will be useful in elucidating the mechanism of germ cell movement. For instance, cytokines have been shown to induce Sertoli cell tight junction disassembly by shutting down the production of tight junction proteins. Other factors such as proteases, protease inhibitors, GTPases, kinases, and phosphatases also come into play. In this review, we focus on this cellular phenomenon, recapping recent developments in the field.

Animals↗
PubMed1979

Leukocyte chemotaxis under agarose: manipulations of serum and plasma before incorporation into agarose can influence cell movement.

In the migration under agarose assay for measurement of polymorphonuclear leukocyte chemotaxis and random migration, the agarose gel ordinarily contains 10% human serum or plasma. Influences of serum and plasma on cell movement using this assay were defined. Utilizing plasma as protein source allowed more random migration and chemotaxis than when serum was used. Heat inactivation of both serum and plasma decreased random migration and chemotaxis. If heating at 56 degrees C was prolonged beyond 15--30 min, leukocyte movement was augmented. Excessive concentrations of platelets or platelet products, or heparin in agarose, also augmented migration, but storage of plasma or sera at various temperatures for 24 h did not appreciably affect migration. Large amounts of hemoglobin in agarose decreased chemotaxis, but the effect of trace amounts of hemolysis was negligible. Handling plasma or sera in standard ways prior to use in the under agarose assay may make this assay more reproducible and sensitive.

Blood↗
PubMed2002

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus.

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.

DNA, Viral↗
PubMed2002

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement.

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

Amino Acid Substitution↗
PubMed1986

Effects of puromycin, cycloheximide and noradrenaline on cell migration within the crypts and on the villi of the small intestine. A model to explain cell movement in both regions.

The normal process of cell migration, occurring as part of the replacement scheme within the small intestinal epithelium, was investigated extensively. The effects of puromycin, cycloheximide and noradrenaline on the movement of tritiated thymidine [( 3H]TdR) prelabelled crypt or villus cells have been studied. These studies have led to the formulation of a model for the mechanism of cell migration, postulating that the crypts and villi behave as separate units, with regard to cell migration, in addition to their distinct structural and functional properties. It is proposed that crypt cell migration is an active process requiring protein synthesis and protein glycosylation, whilst movement of villus epithelial cells is passive, depending on the continued contraction of smooth muscle cells in the lamina propria.

Animals↗