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Intermediate filaments, which make up part of the cytoskeleton, play an important role in providing cells with structural stability [8,3]. The mechanism by which intermediate filaments are able to grow and form networks is, therefore, of interest. Previous mathematical models fit in vitro length distribution data for vimentin (a type of intermediate filament found in cells of mesenchymal origin) only by choosing kinetic rates from obtained from several distinct expressions which arise from various assumptions of the mechanical properties of the filaments. My goal is to improve on existing mathematical models of end-to-end annealing by determining a single underlying expression by which kinetic rates can be determined. Filaments are modeled as semiflexible linear polymers with a probability of annealing which has a decreasing dependence on the length of the filaments. With this approach, the model fit experimental in vitro length distribution data for vimentin [21].

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines – FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.

The C. elegans fibrous organelle (FO) consists of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments (IFs). FOs are most prominent in the epidermis where they overlie body wall muscle and serve to transmit force from the muscle to the cuticle. IFA-2, one of four epidermally expressed IFs whose loss results in epidermal fragility and failure of muscle-cuticle force transmission, localizes to FOs. All IFs contain three domains: a central rod domain and globular head and tail domains. The rod is essential for assembly of IFs into mature filaments while less is known about the functions of the head and tail domains. Roles of the head and tail domains of IFA-2 were examined by expressing GFP-tagged IFA-2 variants in transgenic animals and examining their phenotype and localization in wild-type and null backgrounds. An IFA-2 variant containing only the head domain (IFA-2H) was able to localize to FOs on its own. High levels of larval-stage IFA-2H incorporation into FOs correlate with a dominant-negative muscle detachment phenotype, while low-level incorporation results in healthy animals. This suggests the head domain interacts with FO components essential to tissue integrity. To determine possible interactions between the head domain and FO proteins, the yeast two-hybrid system was used. An initial screen detected that the head and tail domains of IFA-2 are able to interact with each other. To obtain a more complete list of IFA-2H interactors, a C. elegans cDNA library was screened against IFA-2H. After PCR and sequencing of the 239 clones generated from the library screen, 45 unique proteins were found to interact with IFA-2H. Several criteria including functional roles, tissue expression data, and frequency of library inserts has been used to analyze the data and assign relevance to the interactions. One of the proteins found in the library screen, K04G7.1, was investigated further and found to localize throughout the pharynx where it may interact with IFs. This work should help to elucidate the role of the different domains of IFA-2 in FO function and reveal the underlying defects leading to tissue fragility.