Myosin light chain kinase plays an essential role in S. flexneri dissemination.
(1/643)
Shigella flexneri, the causitive agent of bacillary dysentery, has been shown to disseminate in colonic epithelial cells via protrusions that extend from infected cells and are endocytosed by adjacent cells. This phenomenon occurs in the region of the eukaryotic cell's adherens junctions and is inhibited by pharmacological reagents or host cell mutations that completely disrupt the junctional complex. In this study, inhibitors of the myosin light chain kinase (MLCK) were shown to dramatically decrease intercellular spread of S. flexneri but to have no inhibitory effect on bacterial entry, multiplication or actin-based motility within the host cell. Furthermore, cell-to-cell spread of Listeria monocytogenes, another bacterial pathogen that uses an actin-based mechanism to move within the eukaryotic cytoplasm and to spread from cell to cell, was not affected by the MLCK inhibitors, indicating that (1) the inhibition of S. flexneri cell-to-cell spread in treated cells is not due to a complete break down of cell-cell contacts, which was subsequently confirmed by confocal microscopy, and (2) MLCK plays a role in a S. flexneri-specific mechanism of dissemination. Myosin has been shown to play a role in a variety of membrane-based phenomena. The work presented here suggests that activation of this molecule via phosphorylation by MLCK, at the very least participates in the formation of the bacteria-containing protrusion, and could also contribute to the endocytosis of this structure by neighboring cells. (+info)
Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs.
(2/643)
Cells employ a diverse array of signaling mechanisms to establish spatial patterns during development. Nowhere is this better understood than in Drosophila, where the limbs and eyes arise from discrete epithelial sacs called imaginal discs. Molecular-genetic analyses of pattern formation have generally treated discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. Here we demonstrate that during development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development. (+info)
ACAPs are arf6 GTPase-activating proteins that function in the cell periphery.
(3/643)
The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery. (+info)
Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active.
(4/643)
Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved beta3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s). (+info)
Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants.
(5/643)
In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud. (+info)
Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy.
(6/643)
The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase) [1,2]. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50A apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties [3]. Stimulation by EGF resulted in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicated that activated EGFR-CFP interacted with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells. (+info)
Pyramidal cells, patches, and cortical columns: a comparative study of infragranular neurons in TEO, TE, and the superior temporal polysensory area of the macaque monkey.
(7/643)
The basal dendritic arbors of layer III pyramidal neurons are known to vary systematically among primate visual areas. Generally, those in areas associated with "higher" level cortical processing have larger and more spinous dendritic arbors, which may be an important factor for determining function within these areas. Moreover, the tangential area of their arbors are proportional to those of the periodic supragranular patches of intrinsic connections in many different areas. The morphological parameters of both dendritic and axon arbors may be important for the sampling strategies of cells in different cortical areas. However, in visual cortex, intrinsic patches are a feature of supragranular cortex, and are weaker or nonexistent in infragranular cortex. Thus, the systematic variation in the dendritic arbors of pyramidal cells in supragranular cortex may reflect intrinsic axon projections, rather than differences in columnar organization. The present study was aimed at establishing whether cells in the infragranular layers also vary in terms of dendritic morphology among different cortical areas, and whether these variations mirror the ones demonstrated in supragranular cortex. Layer V pyramidal neurons were injected with Lucifer yellow in flat-mounted cortical slices taken from cytoarchitectonic areas TEO and TE and the superior polysensory area (STP) of the macaque monkey. The results demonstrate that cells in STP were larger, had more bifurcations, and were more spinous than those in TE, which in turn were larger, had more bifurcations and were more spinous than those in TEO. These results parallel morphological variation seen in layer III pyramidal neurons, suggesting that increasing complexity of basal dendritic arbors of cells, with progression through higher areas of the temporal lobe, is a general organizational principle. It is proposed that the differences in microcircuitry may contribute to the determination of the functional signatures of neurons in different cortical areas. Furthermore, these results provide evidence that intrinsic circuitry differs across cortical areas, which may be important for theories of columnar processing. (+info)
Synaptically driven calcium transients via nicotinic receptors on somatic spines.
(8/643)
Dendritic spines commonly receive glutamatergic innervation at postsynaptic densities and compartmentalize calcium influx arising from synaptic signaling. Recently, it was shown that a class of nicotinic acetylcholine receptors containing alpha7 subunits is concentrated on somatic spines emanating from chick ciliary ganglion neurons. The receptors have a high relative calcium permeability and contribute importantly to synaptic currents, although they appear to be excluded from postsynaptic densities. Here we show that low-frequency synaptic stimulation of the alpha7-containing receptors induces calcium transients confined to the spines. High-frequency stimulation induces a transient calcium elevation in the spines and a more sustained cell-wide elevation. The high-frequency transient elevation again depends on alpha7-containing receptors, whereas the sustained elevation can be triggered by other nicotinic receptors and depends on calcium release from internal stores and probably influx through voltage-gated L-type calcium channels as well. Retrograde axonal stimulation of the neurons at high frequency mimics synaptic stimulation in producing sustained cell-wide calcium increases that depend on L-type channels and release from internal stores, but it does not produce calcium transients in the spines. Thus frequent action potentials are sufficient to generate the cell-wide increases, but alpha7-containing receptors are needed for spine-specific effects. Patch-clamp recording indicates that alpha7-containing receptors preferentially desensitize at high-frequency stimulation, accounting for the inability of the stimulation to sustain high calcium levels in the spines. The spatial and temporal differences in the patterns of calcium elevation could enable the neurons to monitor their own firing histories for regulatory purposes. (+info)