We have previously reported that Nak1, a group-II germinal center (GC) kinase, is essential for polarized growth in Schizosaccharomyces pombe. Here, we provide evidence that Nak1 regulates cell growth and polarity, in part, through its interactions with Hob1 (an Rvs167/amphiphysin homolog) and Wsp1 (Wiskott-Aldrich-syndrome-protein homolog). We found that Nak1, Hob1 and Wsp1 interact physically, and that both Hob1/green-fluorescent-protein (Hob1-GFP) and Wsp1-GFP fusion proteins localized to F-actin patches at growing cell ends and medial division sites. Hob1-GFP was dissociated from patches in cells lacking Wsp1. Also, Hob1 overexpression dissociated Wsp1-GFP from foci, inhibited Wsp1-directed F-actin formation in vitro and partially restored polarity defects associated with Wsp1 overexpression or nak1 repression. Furthermore, loss of both Wsp1 and Hob1 resulted in rounded cells, slow growth and multiple septae. Together, these observations suggest that Hob1 and Wsp1 cooperate to mediate cell polarity, growth and division. Repression of nak1 resulted in a random redistribution of Hob1-GFP and Wsp1-GFP foci, and inhibition of Wsp1-directed F-actin formation in vitro. Furthermore, hob1Δ and wsp1Δ mutants exhibited synthetic growth defects in combination with nak1 repression, suggesting that Nak1 has redundant functions with Hob1 and Wsp1. Collectively, our results suggest that Nak1 both regulates and cooperates with Hob1 and Wsp1 to promote F-actin formation and polarized cell growth.

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The classical cadherins, definitive proteins of the cadherin superfamily, are characterized functionally by their ability to mediate calcium-dependent cell aggregation in vitro. To test hypothetical mechanisms of adhesion, we have constructed two mutants of the chicken E-cadherin protein, one with the highly conserved HisAla-Val (HAV) sequence motif reversed to Val-Ala-His (VAH), the other lacking the first extracellular domain (EC1). The inversion of HAV to VAH has no effect on the capacity of E-cadherin to mediate adhesion. Deletion of EC1 completely eliminates the ability of E-cadherin to mediate homophilic adhesion, but the deletion mutant is capable of adhering heterophilically to both unmutated E-cadherin and to the HAV/VAH mutant. These results demonstrate that the conserved HAV sequence motif is not involved in cadherin-mediated adhesion as has been suggested previously and supports the idea that in the context of the cell surface, cadherin-mediated cell-cell adhesion involves an interaction of EC1 with other domains of the cadherin extracellular moiety and not the "linear zipper" model, which posits trans interactions only between EC1 on apposing cell surfaces.Classical cadherins were defined initially by their ability to mediate calcium-dependent cell-cell adhesion (1-5). Both the extracellular and the intracellular portions of the cadherin play critical roles in mediating adhesion (6). Perturbation and expression experiments have demonstrated that cadherin-mediated adhesion promotes a range of cellular processes subsequent to adhesion, including epithelial polarization (7, 8), blastula compaction (2), neurite outgrowth (9, 10), and formation of desmosomes (11, 12) and gap junctions (13-16).Classical cadherin proteins at the cell surface consist of five extracellular domains (ECs), 1 a transmembrane sequence, and a cytoplasmic domain (Fig. 1). Each cadherin extracellular domain shares a folding topology of seven ␤-strands arranged in two ␤-sheets forming a barrel with hydrophobic amino acid side chains largely sequestered within the interior (17-20). The amino and carboxyl termini emerge from opposite ends of each folded domain, maintaining an orientation with the amino terminus directed away from the cell. Conserved amino acid residues that are capable of coordinating calcium ions are present at the ends of each barrel (17)(18)(19)(20). Binding of calcium at these sites maintains the structural integrity of the cadherin ECs, giving the protein a rigid conformation that allows it to mediate cell-cell adhesion. A single amino acid substitution in a calcium binding site between EC1 and EC2 or between EC2 and EC3 can abolish adhesion and increase motility, whereas mutations in other calcium-binding elements do not affect these behaviors (21,22).Cadherins expressed on adjacent cells homoassociate through an antiparallel, trans, interaction of their extracellular regions. The bonds that they form across the intercellular space hold the plasma membranes of adjacent cells in close proximity. Adhesive strength...

A major challenge in effectively treating infections is to provide timely diagnosis of a bacterial or viral agent. Current cell culture methods require >24 h to identify the cause of infection. The Toll-like Receptor (TLR) family of proteins can identify classes of pathogens and has been shown to work well in an impedance-based biosensor, where the protein is attached to an electrode via a self-assembled monolayer (SAM). While the sensitivity of these sensors has been good, they contain a high resistance (>1 kΩ) SAM, generating relatively small signals and requiring longer data collection, which is ill-suited to implementation outside of a laboratory. Here, we describe a novel approach to increase the signal magnitude and decrease the measurement time of a TLR-4 biosensor by inserting a redox-active ferrocenyl-terminated alkanethiol into a mixed SAM containing hydroxyl- and carboxyl-terminated alkanethiols. The SAM formation and modification was confirmed via contact angle and X-ray photoelectron spectroscopy measurements, with TLR-4 immobilization demonstrated through a modified immunosorbent assay. It is shown that these TLR-4 biosensors respond selectively to their intended target, Gram-negative bacteria at levels between 1 and 105 lysed cells/mL, while remaining insensitive to Gram-positive bacteria or viral particles at up to 105 particles/mL. Furthermore, the signal enhancement due to the addition of ferrocene decreased the measurement time to less than 1 min and has enabled this sensor to be used with an inexpensive, portable, hand-held potentiostat that could be easily implemented in field settings.