The F-BAR family of proteins play important roles in many cellular processes by regulating both membrane and actin dynamics. The CIP4 family of F-BAR proteins is widely recognized to function in endocytosis by elongating endocytosing vesicles. However, in primary cortical neurons, CIP4 concentrates at the tips of extending lamellipodia and filopodia and inhibits neurite outgrowth. Here, we report that the highly homologous CIP4 family member, FBP17, induces tubular structures in primary cortical neurons and results in precocious neurite formation. Through domain swapping and deletion experiments, we demonstrate that a novel polybasic region between the F-BAR and HR1 domains is required for membrane bending. Moreover, the presence of a poly-PxxP region in longer splice isoforms of CIP4 and FBP17 largely reverses the localization and function of these proteins. Thus, CIP4 and FBP17 function as an antagonistic pair to fine-tune membrane protrusion, endocytosis, and neurite formation during early neuronal development.
In utero electroporation (IUE) is a powerful tool for testing the role of genes in neuronal migration and function, but this technique suffers from high degrees of variability. Such variability can result from inconsistent surgery, developmental gradients along both rostral-caudal and medial-lateral axes, differences within littermates and from one litter to another. Comparisons between control and experimental electroporations rely on section matching, which is inherently subjective. These sources of variability are cumulative, leading to difficult to interpret data and an increased risk of both false positives and false negatives. To address these limitations, we developed two tools: (1) a new plasmid, termed Double UP, which combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls, and (2) an automated program for unbiased and precise quantification of migration. In concert, these tools allow for more rigorous and objective experiments, while decreasing the mice, time, and reagents required to complete studies.
In Utero Electroporation is a powerful tool for testing the role of genes in neuronal migration, but this technique suffers from high degrees of variability due to multiple factors. Therefore, we developed Double UP, a novel approach that combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls. This technique allows for more rigorous quantification of migration, while decreasing the number of animals, reagents and time to complete experiments.
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