Person:

Lian, Gewei

Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB) gene develop spondylocarpotarsal syndrome (SCT), a disorder characterized by dwarfism (delayed bone formation) and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation). To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB−/− mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1+/Col10a1+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression) and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation) mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins.

The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and cytokinesis and differentiation. These developmental processes can be regulated through actin dependent vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes and proteins. Disruption in the actin cytoskeleton in the brain gives rise to periventricular heterotopia (PH), a malformation of cortical development, characterized by abnormal neurons clustered deep in the brain along the lateral ventricles. This disorder can give rise to seizures, dyslexia and psychiatric disturbances. Anatomically, PH is characterized by a smaller brain (impaired proliferation), heterotopia (impaired initial migration) and disruption along the neuroependymal lining (impaired cell-cell adhesion). Genes causal for PH have also been implicated in actin-dependent processes. The current review provides mechanistic insight into actin cytoskeletal regulation of cortical development in the context of this malformation of cortical development.

The effects of actin dependent molecular mechanisms in coordinating cellular proliferation, migration and differentiation during embryogenesis are not well-understood. We have previously shown that actin-binding Filamin A (FlnA) and actin-nucleating Formin 2 (Fmn2) influence the development of the brain causing microcephaly in mice. In this study, we broaden this phenotype to explore the effects of these two proteins in the development of extra-CNS organ systems, including the gut, muscle, and skeleton. We observed defects in rib and sternum midline closure leading to thoracoabdominal schisis in FlnA+Fmn2 knockout mice, reminiscent of the pentalogy of Cantrell syndrome. These mice exhibit shortened guts, as well as thinned thoracic muscle mass. Immunostaining showed these changes are partially caused by a decrease in the number of presumptive mesenchymal proliferating cells with loss of either FlnA or FlnA+Fmn2. This proliferation defect appears to be in part due to delayed differentiation in these regions. While both FlnA and FlnA+Fmn2 mice show reduced cell death relative to WT control, increased caspase staining was seen in the double null relative to FlnA null suggesting that this could also contribute to the FlnA+Fmn2 phenotype. Therefore FlnA and Fmn2 are likely essential to cell proliferation, differentiation and cell death in a variety of tissues and organs, further reiterating the importance of vesicle trafficking in regulation of development.