Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis

GCP6 Binds to Intermediate Filaments: A Novel Function of Keratins in the Organization of Microtubules in Epithelial Cells

In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the γ-tubulin ring complex, γ-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in γ-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced γ-tubulin from the cytoskeleton and resulted in depolarization of γ-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia

Bioceramics composition modulate resorption of human osteoclasts

Biomaterials used in bone regeneration are designed to be gradually resorbed by the osteoclast and replaced by new bone formed through osteoblastic activity. The aim of the present study is to analyze the role of osteoclasts in the resorption process. The attachment of human osteoclasts and the appearance of their resorption lacunae, when cultured on either the resorbable crystalline, calcium orthophosphate materials or on the long-term stable bioceramic material was investigated. The resorbable materials contain Ca10[K,Na](PO4)7 (AW-Si) and Ca2KNa(PO4)2 (GB14, GB9 & D9/25) as their main crystal phases, however they differ in their total solubility. These differences result from small variations in the composition. The long-term stable material consist of about 30% fluorapatite beside calcium zirconium phosphate (Ca5(PO4)3F + CaZr4(PO4)6) and shows a very small solubility. AW-Si has an alkali containing crystalline phase, Ca10[K,Na](PO4). While GB14, GB9 and D9/25 contain the crystalline phase Ca2KNa(PO4)2 with small additions of crystalline and amorphous diphosphates and/or magnesium potassium phosphate (GB14). D9/25 and AW-Si is less soluble compared to GB14, and GB9 among the resorbable materials. Resorbable and long-term stable materials vary in their chemical compositions, solubility, and surface morphology. Osteoclasts modified the surface in their attempts to resorb the materials irrespective of the differences in their physical and chemical properties. The depth and morphology of the resorption imprints were different depending on the type of material. These changes in the surface structure created by osteoclasts are likely to affect the way osteoblasts interact with the materials and how bone is subsequently formed.Y. Ramaswamy, D. R. Haynes, G. Berger, R. Gildenhaar, H. Lucas, C. Holding and H. Zreiqa