The C. elegans rab family: identification, classification and toolkit construction.

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).

Mechanosensory neurite termination and tiling depend on SAX-2 and the SAX-1 kinase.

Mechanosensory neurons provide accurate information about stimulus location by restricting their sensory dendrites to nonoverlapping regions, a pattern called tiling. Here, we show that C. elegans sax-1 and sax-2 regulate mechanosensory tiling by controlling the termination point of sensory dendrites. During development, the posterior PLM mechanosensory dendrite overlaps transiently with the anterior ALM mechanosensory neuron. This overlap is eliminated during a discrete period of paused or slowed PLM process growth, between an early period of rapid outgrowth and a later period of maintenance growth. In sax-2 mutants, the PLM sensory dendrite fails to slow between the active growth and maintenance growth phases, leading to sustained overlap of anterior and posterior mechanosensory processes. sax-2 encodes a large conserved protein with HEAT/Armadillo repeats that functions with sax-1, an NDR cell morphology-regulating kinase. High-level expression of sax-2 leads to premature neurite termination, suggesting that SAX-2 can directly inhibit neurite growth.