genetic-manipulation-cerebellar-granule-neurons-vitro-vivo-to-study

Genetic Manipulation of Cerebellar Granule Neurons <em>In Vitro</em> and <em>In Vivo</em> to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Max Planck Institute of Experimental Medicine

Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB)

The fluorescence emission spectrum of N-acetyl tryptophan amide (NATA) in 20 mM K-phosphate buffer, pH 7.5, with excitation at 295 nm, when subjected to second derivatization, showed two troughs at 340 1.0 nm (A) and 358.5 1.0 nm (B). Linear dependence of derivative intensities at A and B was observed with increasing NATA concentration between 0-30 nM but the intensity ratio (B/A), termed R, was found to be invariant at 0.70 0.05. R remained unaffected with variation of the pH (4-10), temperature (15-70 degrees C), salt concentration (0-2 M NaCl), and excitation wavelength between 280-300 nm. A 50-fold molar excess of N-acetyl tyrosine over 10 nM NATA and inclusion of a quencher like 0.8 M acrylamide, 0.4 M potassium iodide or trichloroethanol had no effect on R. It was, however, linearly dependent on the polarity of the solvent-in 1,4-dioxane it became 0.07 0.05. Derivative spectra of tryptophans of proteins largely resembled that of NATA. Low R values of between 0.02-0.34 were observed for proteins under native conditions, which is consistent with the general buried character of tryptophan residues. R increased to 0.6-0.9 after unfolding with denaturants or extensive proteolysis and decreased to close to the original value after refolding. The equilibrium unfolding transitions of proteins expressed as R largely resembled the transitions measured using other physical parameters. R appears to be a more sensitive index for monitoring the hydrophobic environment of tryptophans in protein compared to parameters like emission maxima or intensity of underivatised spectra.

Second derivative fluorescence spectra of indole compounds.

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Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

Chaitali Mukherjee

Cellular and Molelcular Neurobiology

Max Planck Institute of Experimental Medicine

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Chaitali Mukherjee

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Max Planck Institute of Experimental Medicine

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Cellular and Molelcular Neurobiology