Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

Vesicle formation and endocytosis: function, machinery, mechanisms, and modeling.

Vesicle formation provides a means of cellular entry for extracellular substances and for recycling of membrane constituents. Mechanisms governing the two primary endocytic pathways (i.e., caveolae- and clathrin-mediated endocytosis, as well as newly emerging vesicular pathways) have become the focus of intense investigation to improve our understanding of nutrient, hormone, and drug delivery, as well as opportunistic invasion of pathogens. In this review of endocytosis, we broadly discuss the structural and signaling proteins that compose the molecular machinery governing endocytic vesicle formation (budding, invagination, and fission from the membrane), with some regard for the specificity observed in certain cell types and species. Important biochemical functions of endocytosis and diseases caused by their disruption also are discussed, along with the structures of key components of endocytic pathways and their known mechanistic contributions. The mechanisms by which principal components of the endocytic machinery are recruited to the plasma membrane, where they interact to induce vesicle formation, are discussed, together with computational approaches used to simulate simplified versions of endocytosis with the hope of clarifying aspects of vesicle formation that may be difficult to determine experimentally. Finally, we pose several unanswered questions intended to stimulate further research interest in the cell biology and modeling of endocytosis.

Microtubule-targeting-dependent reorganization of filopodia.

Interaction between the microtubule system and actin cytoskeleton has emerged as a fundamental process required for spatial regulation of cell protrusion and retraction activities. In our current studies, analysis of digital fluorescence images revealed targeting of microtubules to filopodia in B16F1 melanoma cells and fibroblasts. We investigated the functional consequence of targeting on filopodia reorganization and examined mechanisms by which microtubules may be guided to, or interact with, filopodia. Live cell imaging studies show that targeting events in lamellipodia wings temporally correlated with filopodia turning toward the lamellipodium midline and with filopodia merging. Rapid uncoupling of targeting with nocodazole decreased filopodia merging events and increased filopodia density. Total internal reflection fluorescence microscopy identified microtubules near the ventral surface and upward movement of targeted filopodia. The role of adhesion sites and microtubule plus-end proteins in targeting was investigated. Correlation of adhesion sites with microtubule targeting to filopodia was not observed and depletion of microtubule plus-end proteins did not significantly alter targeting frequency. We propose that microtubules target filopodia, independent of focal adhesions and plus-end proteins, causing filopodia movement and microtubules regulate filopodia density in lamellipodia wings through filopodia merging events.

Kinetic-structural analysis of neuronal growth cone veil motility.

Neuronal growth cone advance was investigated by correlative light and electron microscopy carried out on chick dorsal root ganglion cells. Advance was analyzed in terms of the two principal organelles responsible for protrusive motility in the growth cone - namely, veils and filopodia. Veils alternated between rapid phases of protrusion and retraction. Electron microscopy revealed characteristic structural differences between the phases. Our results provide a significant advance in three respects: first, protruding veils are comprised of a densely branched network of actin filaments that is lamellipodial in appearance and includes the Arp2/3 complex. On the basis of this structural and biomarker evidence, we infer that the dendritic nucleation and/or array-treadmilling mechanism of protrusive motility is conserved in veil protrusion of growth cones as in the motility of fibroblasts; second, retracting veils lack dendritic organization but contain a sparse network of long filaments; and third, growth cone filopodia have the capacity to nucleate dendritic networks along their length, a property consistent with veil formation seen at the light microscopic level but not previously understood in supramolecular terms. These elements of veil and filopodial organization, when taken together, provide a conceptual framework for understanding the structural basis of growth cone advance.

Lamellipodial actin mechanically links myosin activity with adhesion-site formation.

Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.

Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility.

Spotted fever group Rickettsia are obligate intracellular pathogens that exploit the host cell actin cytoskeleton to promote motility and cell-to-cell spread. Although other pathogens such as Listeria monocytogenes use an Arp2/3 complex-dependent nucleation mechanism to generate comet tails consisting of Y-branched filament arrays, Rickettsia polymerize tails consisting of unbranched filaments by a previously unknown mechanism. We identified genes in several Rickettsia species encoding proteins (termed RickA) with similarity to the WASP family of Arp2/3-complex activators. Rickettsia rickettsii RickA activated both the nucleation and Y-branching activities of the Arp2/3 complex like other WASP-family proteins, and was sufficient to direct the motility of microscopic beads in cell extracts. Actin tails generated by RickA-coated beads consisted of Y-branched filament networks. These data suggest that Rickettsia use an Arp2/3 complex-dependent actin-nucleation mechanism similar to that of other pathogens. We propose that additional Rickettsia or host factors reorganize the Y-branched networks into parallel arrays in a manner similar to a recently proposed model of filopodia formation.

Orientational order of the lamellipodial actin network as demonstrated in living motile cells.

Lamellipodia of crawling cells represent both the motor for cell advance and the primary building site for the actin cytoskeleton. The organization of actin in the lamellipodium reflects actin dynamics and is of critical importance for the mechanism of cell motility. In previous structural studies, the lamellipodial actin network was analyzed primarily by electron microscopy (EM). An understanding of lamellipodial organization would benefit significantly if the EM data were complemented and put into a kinetic context by establishing correspondence with structural features observable at the light microscopic level in living cells. Here, we use an enhanced phase contrast microscopy technique to visualize an apparent long-range diagonal actin meshwork in the advancing lamellipodia of living cells. Visualization of this meshwork permitted a correlative light and electron microscopic approach that validated the underlying organization of lamellipodia. The linear features in the light microscopic meshwork corresponded to regions of greater actin filament density. Orientation of features was analyzed quantitatively and compared with the orientation of actin filaments at the EM level. We infer that the light microscopic meshwork reflects the orientational order of actin filaments which, in turn, is related to their branching angle.

Mechanism of filopodia initiation by reorganization of a dendritic network.

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Lambda-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

Heart and the peritoneal cover of the gut sinus in the polychaete Arenicola marina: an ultrastructural and autoradiographic study.

To elucidate the cellular mechanism underlying the growth of the peritoneal cover of the gut sinus and the heart in the polychaete Arenicola marina, cellular organization of these structures and proliferative potential of their cells were investigated using electron microscopy and electron microscopic autoradiography. Arenicola has a pair of dorsolaterally situated hearts connected to the gut sinus via a short duct and composed of two muscular layers separated by a layer of the extracellular matrix (ECM). The peritoneal cover of the gut sinus and the outer muscular layer of the heart present a myoepithelial layer resting on the ECM. The inner muscular layer of the heart is composed of myofibril-containing cells lacking well-defined polarity in arrangement of organelles. However, their persistent connection to branches of the ECM and the adherens-like intercellular junctions allow for considering the inner layer a modified myoepithelium. In the peritoneal cover of the gut sinus and in both myoepithelial layers of the heart, noncontractile epithelial cells have been observed. As determined by thymidine labeling, these epithelial cells are capable of DNA synthesis, while myoepithelial cells are not. Some suggestions are made about the myogenic nature of the epithelial cells in the investigated structures of A. marina.