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To date, the functions of most neural intermediate filament (IF) proteins have remained elusive. Peripherin is a type III intermediate filament (IF)protein that is expressed in developing and in differentiated neurons of the peripheral and enteric nervous systems. It is also the major IF protein expressed in PC12 cells, a widely used model for studies of peripheral neurons. Dramatic increases in peripherin expression have been shown to coincide with the initiation and outgrowth of axons during development and regeneration, suggesting that peripherin plays an important role in axon formation. Recently, small interfering RNAs (siRNA) have provided efficient ways to deplete specific proteins within mammalian cells. In this study, it has been found that peripherin-siRNA depletes peripherin and inhibits the initiation, extension, and maintenance of neurites in PC12 cells. Furthermore, the results of these experiments demonstrate that peripherin IF are critical determinants of the overall shape and architecture of neurons. INTRODUCTIONThe cytoskeleton of vertebrate cells consists of three major types of protein networks: intermediate filaments (IF), microfilaments (MF), and microtubules (MT). Intermediate filaments are the most diverse of the three because they are encoded by Ͼ65 genes, making the IF superfamily one of the 100 largest in the human genome (Hesse et al., 2001). These genes are developmentally regulated, resulting in the cell type-specific expression of IF. This is clearly evident in the nervous system, where at least seven different IF proteins are expressed, ranging from the complex neurofilament (NF) heteropolymers composed of the type IV triplet proteins NF-L, NF-M, and NF-H, to the simpler homopolymers of the type III IF protein peripherin (Leung et al., 1998). Peripherin forms the major IF system of peripheral and enteric neurons, and it is abundantly expressed in PC12 cells, a widely used model for studies of peripheral neurons (Parysek and Goldman, 1987;Leonard et al., 1988; Portier et al., 1983a,b).One of the hallmarks of mature or terminally differentiated neurons is their remarkable shape, highlighted by extremely long cytoplasmic processes such as axons. The initiation, extension, and maintenance of axons involve the coordinated interactions of different cytoskeletal proteins (Mueller, 1999;Dickson, 2002). To date, only MT and MF have been considered essential for growth cone activity and axon outgrowth (Letourneau, 1996). In contrast, the contribution of neural IF to these processes has not been defined.The expression patterns of neural IF are highly correlated with different phases of axonal development. Type III IF, such as peripherin and vimentin, are present throughout early stages of outgrowth, and later type IV NF triplet proteins are expressed as axons reach maturity (Cochard and Paulin, 1984;Troy et al., 1990a). In mature neurons, NF appear to be major determinants of axon caliber, and thus conduction velocity (Lasek et al., 1983;Hoffman et al., 1987). However, little is known about ...

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (⌬vsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the ⌬vsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the ⌬vsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.Vibrionaceae are a predominant family of gram-negative bacteria found in aquatic environments (30). Bacteria within this family demonstrate a high degree of genetic diversity and are able to colonize very different types of niches. They live freely as planktonic forms in the water column or are associated in biofilms or with host organisms as pathogenic, commensal, or mutualistic bacteria. To date, eight genome sequences from Vibrionaceae have been made available: those of Vibrio cholerae strains N16961 and 0395, V. parahaemolyticus RIMD2210633, V. vulnificus strains YJ016 and CMCP6, V. fischeri ES114, V. harveyi ATCC BAA-1116, and Photobacterium profundum SS9 (3,10,19,24,32). More recently, we have completed the sequencing of the genome of V. splendidus strain LGP32, an oyster (Crassostrea gigas) pathogen