The Cofilin/Limk1 Pathway Controls the Growth Rate of Both Developing and Regenerating Motor Axons.

Regenerating axons often have to grow considerable distances to reestablish circuits, making functional recovery a lengthy process. One solution to this problem would be to co-opt the "temporal" guidance mechanisms that control the rate of axon growth during development to accelerate the rate at which nerves regenerate in adults. We have previously found that the loss of Limk1, a negative regulator of cofilin, accelerates the rate of spinal commissural axon growth. Here, we use mouse models to show that spinal motor axon outgrowth is similarly promoted by the loss of Limk1, suggesting that temporal guidance mechanisms are widely used during development. Furthermore, we find that the regulation of cofilin activity is an acute response to nerve injury in the peripheral nervous system. Within hours of a sciatic nerve injury, the level of phosphorylated cofilin dramatically increases at the lesion site, in a Limk1-dependent manner. This response may be a major constraint on the rate of peripheral nerve regeneration. Proof-of-principle experiments show that elevating cofilin activity, through the loss of Limk1, results in faster sciatic nerve growth, and improved recovery of some sensory and motor function.SIGNIFICANCE STATEMENT The studies shed light on an endogenous, shared mechanism that controls the rate at which developing and regenerating axons grow. An understanding of these mechanisms is key for developing therapies to reduce painful recovery times for nerve-injury patients, by accelerating the rate at which damaged nerves reconnect with their synaptic targets.

Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.

Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos.

During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Gα-GPR-LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.