Focal Defects in Single-Celled Tubes Mutant for Cerebral Cavernous Malformation 3, GCKIII, or NSF2

Abstract

Tubes of differing cellular architecture connect into networks. In the Drosophila tracheal system, two tube types connect within single cells (terminal cells); however, the genes that mediate this interconnection are unknown. Here we characterize two genes that are essential for this process: lotus, required for maintaining a connection between the tubes, and wheezy, required to prevent local tube dilation. We find that lotus encodes N-ethylmaleimide Sensitive Factor 2 (NSF2), while wheezy encodes Germinal Center Kinase III (GCKIII). GCKIIIs are effectors of Cerebral Cavernous Malformation 3 (CCM3), a protein mutated in vascular disease. Depletion of CCM3 by RNA interference phenocopies wheezy; thus, CCM3 and GCKIII, which prevent capillary dilation in humans, prevent tube dilation in Drosophila trachea. Ectopic junctional and apical proteins are present in wheezy terminal cells, and we show that tube dilation is suppressed by reduction of NSF2, of the apical determinant Crumbs, or of septate junction protein Varicose.

Compensatory branching morphogenesis of stalk cells in the Drosophila trachea

“…A fraction of control terminal cells (15%, n=84) had a nucleus-proximal autocellular tube (<4 μm in length; supplementary material Fig. S1A), as previously described for embryonic (Samakovlis et al, 1996) and larval (Song et al, 2013) terminal cells. A comparable fraction of okg (18%, n=101) and cnj (25%, n=72) terminal cells had similar segments of short unbranched autocellular tube ( Fig.…”

“…The latter seemed likely, as seamed tubes within terminal cells appeared to terminate at internal intercellular junctions. However, the organization of seamed-to-seamless tube transitions remains unclear for terminal cells: some evidence suggests the connection of seamed and seamless tubes within a terminal cell 'transition zone' (Samakovlis et al, 1996;Song et al, 2013), whereas other data suggest that terminal cells make exclusively seamless tubes ( 2003). At wild-type stalk cell-fusion cell interfaces, the stalk cell extends a seamed tube into the fusion cell like "a finger poking into a balloon" (Uv, 2003) -note that these 'fingers' end in ring-like intercellular junctions; this has led to the suggestion that terminal cell autocellular tubes might also be stalk cell 'fingers'.…”

“…1A,B) (Ghabrial et al, 2003;Samakovlis et al, 1996). Prior work (Song et al, 2013) suggested that connections between tubes of different architectures requires ongoing maintenance, but the maintenance program is not well understood. Here we characterize mutants with late onset connection defects and uncover an unappreciated ability of autocellular seamed tubes to undergo compensatory growth and branching when terminal cells are unable to expand their apical membrane domain.…”

Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae

“…The localization of SvkA is also similar to Sps1 in that both have cytonuclear localization and enrichment at the membrane when cytokinesis occurs (Rohlfs et al 2007), suggesting that Sps1 and related GCKIII kinases may have primordial origins in stress response and influencing cytokinesis in these contexts. Similarly, the Drosophila GCKIII kinase Wheezy has been shown to be involved in trachea development, where wheezy mutants have inappropriate trafficking of membranebound adhesion proteins and increase accumulation of Crumbs protein on the tracheal membrane (Song et al 2013), although Crumbs and Ssp1 do not show any obvious homology.…”

“…The localization of SvkA is also similar to Sps1 in that both have cytonuclear localization and enrichment at the membrane when cytokinesis occurs (Rohlfs et al 2007), suggesting that Sps1 and related GCKIII kinases may have primordial origins in stress response and influencing cytokinesis in these contexts. Similarly, the Drosophila GCKIII kinase Wheezy has been shown to be involved in trachea development, where wheezy mutants have inappropriate trafficking of membranebound adhesion proteins and increase accumulation of Crumbs protein on the tracheal membrane (Song et al 2013), although Crumbs and Ssp1 do not show any obvious homology.The timing of prospore membrane closure, as with any cytokinetic event, needs to be properly regulated. The APC/C is a highly regulated complex with direct roles regulating the events that occur during the meiotic division and subject to complex feedback loops (Cooper et al 2000;Oelschlaegel et al 2005;Penkner et al 2005;Tan et al 2010;Tsuchiya et al 2011;Okaz et al 2012).…”