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Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization. [ABSTRACT FROM AUTHOR]

The functions of long smooth muscle myosin light chain kinase (L-MLCK), a molecule with multiple domains, are poorly understood. To examine the existence of further potentially functional domains in this molecule, we analyzed its amino acid sequence with atango program and found a putative aggregation domain located at the 4Ig domain of the N-terminal extension. To verify its aggregation capability in vitro, expressible truncated L-MLCK variants driven by a cytomegalovirus promoter were transfected into cells. As anticipated, only the overexpression of the 4Ig fragment led to particle formation in Colon26 cells. These particles contained 4Ig polymers and actin. Analysis with detergents demonstrated that the particles shared features in common with aggregates. Thus, we conclude that the 4Ig domain has a potent aggregation ability. To further examine this aggregation domain in vivo, eight transgenic mouse lines expressing the 4Ig domain (4Ig lines) were generated. The results showed that the transgenic mice had typical aggregation in the thigh and diaphragm muscles. Histological examination showed that 7.70 ± 1.86% of extensor digitorum longus myofibrils displayed aggregates with a 36.44% reduction in myofibril diameter, whereas 65.13 ± 3.42% of diaphragm myofibrils displayed aggregates and the myofibril diameter was reduced by 43.08%. Electron microscopy examination suggested that the aggregates were deposited at the mitochondria, resulting in structural impairment. As a consequence, the oxygen consumption of mitochondria in the affected muscles was also reduced. Macrophenotypic analysis showed the presence of muscular degeneration characterized by a reduction in force development, faster fatigue, decreased myofibril diameters, and structural alterations. In summary, our study revealed the existence of a novel aggregation domain in L-MLCK and provided a direct link between L-MLCK and aggregation. The possible significance and mechanism underlying the aggregation-based pathological processes mediated by L-MLCK are also discussed. [ABSTRACT FROM AUTHOR]