Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes.

Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type.

Haemostatic role of intermediate filaments in adhered platelets: importance of the membranous system stability.

The role of platelets in coagulation and the haemostatic process was initially suggested two centuries ago, and under appropriate physiological stimuli, these undergo abrupt morphological changes, attaching and spreading on damaged endothelium, preventing bleeding. During the adhesion process, platelet cytoskeleton reorganizes generating compartments in which actin filaments, microtubules, and associated proteins are arranged in characteristic patterns mediating crucial events, such as centralization of their organelles, secretion of granule contents, aggregation with one another to form a haemostatic plug, and retraction of these aggregates. However, the role of Intermediate filaments during the platelet adhesion process has not been explored. J. Cell. Biochem. 114: 2050-2060, 2013. © 2013 Wiley Periodicals, Inc.

Extensive rod and cone photoreceptor-cell degeneration in rat models of giant axonal neuropathy: implications for gene therapy of human disease.

Background: Giant axonal neuropathy (GAN; ORPHA: 643; OMIM# 256850) is a rare, hereditary, pediatric neurodegenerative disorder associated with intracellular accumulations of intermediate filaments (IFs). Validation of therapeutic efficacy and viral vector delivery systems with GAN knockout (KO) mouse models has provided the springboard for the development of a viral vector being delivered intrathecally in an ongoing Phase I gene therapy clinical trial for the treatment of children with GAN (https://clinicaltrials.gov/ct2/show/NCT02362438).Purpose: To characterize the ocular pathologic phenotype of newly developed GAN rat models.Materials and Methods: Microscopic examination of eyes at various timepoints.Results: We noted the unexpected finding of progressive and extensive degeneration of rod and cone photoreceptor (PR) cells in the retinas of GAN rat models.Conclusion: This PR-cell loss in rat models of GAN raises the possibility that PR-cell loss may contribute to the visual impairment observed in human GAN. The intrathecal viral vector employed in the ongoing Phase I gene therapy clinical trial for the treatment of children with GAN was not specifically designed to address PR-cell degeneration. If GAN-associated PR-cell loss is present and clinically significant in humans, then future treatment protocols for GAN may need to include a gene transfer approach or combinatorial treatment strategy that also targets retinal PR cells.

Advancing the pathologic phenotype of giant axonal neuropathy: early involvement of the ocular lens.

Giant axonal neuropathy (GAN; ORPHA: 643; OMIM# 256850) is a rare, hereditary, pediatric neurodegenerative disorder associated with intracellular accumulations of intermediate filaments (IFs). GAN knockout (KO) mouse models mirror the IF dysregulation and widespread nervous system pathology seen in human GAN. Validation of therapeutic efficacy and viral vector delivery systems with these GAN KO models has provided the springboard for the development of a viral vector being delivered intrathecally in an ongoing Phase I gene therapy clinical trial for the treatment of children with GAN ( https://clinicaltrials.gov/ct2/show/NCT02362438 ). During the course of a comprehensive pathologic characterization of the GAN KO mouse, we discovered the very early and unexpected involvement of the ocular lens. Light microscopy revealed the presence of intracytoplasmic inclusion bodies within lens epithelial cells. The inclusion bodies showed strong immunohistochemical positivity for glial fibrillary acidic protein (GFAP). We confirmed that intracytoplasmic inclusion bodies are also present within lens epithelial cells in human GAN. These IF inclusion bodies in lens epithelial cells are unique to GAN. Similar IF inclusion bodies in lens epithelial cells have not been reported previously in experimental animal models or human diseases. Since current paradigms in drug discovery and drug repurposing for IF-associated disorders are often hindered by lack of validated targets, our findings suggest that lens epithelial cells in the GAN KO mouse may provide a potential target, in vivo and in vitro, for evaluating drug efficacy and alternative therapeutic approaches in promoting the clearance of IF inclusions in GAN and other diseases characterized by intracellular IF accumulations.

Epithelial sodium channel modulates platelet collagen activation.

Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and ß-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation.