Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.
information on 85 patients in the 25 families studied, although there were approximately 144 affected individuals across four or fi ve generations in 60% of the families. The mean number of affected individuals per family was 5.7. Of the 30 families, fi ve had mutations in SOD1 (20), and mutations in FUS/TLS were identifi ed in two.Family F-19. The proband (IV:18) presented at 41 years of age for genetic screening. She had developed progressive weakness and wasting of the upper limbs (UL) over the preceding 11 months. Brain and cervical MRI were normal. EMG revealed active generalized denervation. Clinical examination revealed diffuse fasciculations and marked scapulohumeral amyotrophy in both arms. Six months later, proximal muscle involvement had worsened, with dropped head and predominant lower motor neuron (LMN) signs. Respiratory failure developed 25 months after clinical onset, and death occurred six months later.Her brother (IV:16) had died of ALS at 28 years of age, after a rapidly progressive (10 months) Patients and methods PatientsThe mean age of our FALS patient cohort was 49.3 (SD Ϯ 12.0) years. We had suffi cient clinical AbstractOur objective was to investigate the prevalence of FUS/TLS mutations in a Catalan familial ALS cohort undergoing a mutational study for SOD1 in 2006. We screened 25 probands from non-SOD1 families for FUS/TLS mutations. We identifi ed two FALS probands with FUS/TLS mutations. One carried a C-to-T transition at nucleotide position 1561 (c.1561C Ͼ T) producing a p.R521C sequence change at protein level. The phenotype was characterized by a young age at onset (38.2 years old), proximal limb girdle weakness, predominant lower motor neuron signs and dropped head. Survival time ranged from 10 to 36 months. Obligate asymptomatic carriers were detected. Our second ALS6 pedigree carried a C-to-T transition at nucleotide position 1528 (c.1528G Ͼ A) producing a p.K510E sequence change at protein level. The phenotype was of an early onset ( Ͻ 40 years old), predominant lower motor neuron disease with short survival (nine months). In conclusion, these are the fi rst two FUS/TLS mutations identifi ed in Spain. The prevalence of this form of FALS (8%) is similar to the Dutch and British populations. FUS/TLS mutations are the second most common cause of FALS in our population.
We evaluated a possible genotype-phenotype correlation and looked for a founder effect in four Mediterranean families carrying the I112M SOD1 mutation. The structural characteristics of the mutated protein were also analysed. Clinical data of FALS subjects from four families were evaluated. Mutational analysis of the SOD1 gene was carried out by direct sequencing. A haplotype study was carried out using 11 polymorphic markers flanking the SOD1 gene. Structural analysis was performed by means of homology modelling and molecular graphics methods. The clinical pattern of 17 FALS patients was characterized by prevalent spinal onset, mean age at onset of 47.1 years and mean duration of 20.7 months. Several obligate carriers were observed. These findings indicate that the I112M mutation is consistently associated with a uniform, fast-progressing phenotype with reduced penetrance of the disease. The haplotype analysis did not show a common haplotype among the Spanish families and the Italian family; however, a possible common founder could be hypothesized for Spanish families. From a structural viewpoint, mutation at codon 112 seems to confer a severe phenotype, probably related to altered protein functionality.
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