Peptidases compartmentalized to the Ascaris suum intestinal lumen and apical intestinal membrane

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine

Rab35 regulates evoked exocytosis of endothelial Weibel-Palade bodies

Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exoyctosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling haemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15 and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase-activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target RabGTPases. Among the inhibitory RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von-Willebrand factor (VWF) and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat and PH domain-containing protein ACAP2 as Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6 supported histamine-evoked WPB exocytosis as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6

Rab35 regulates evoked exocytosis of endothelial Weibel-Palade bodies

Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exoyctosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling haemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15 and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase-activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target RabGTPases. Among the inhibitory RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von-Willebrand factor (VWF) and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat and PH domain-containing protein ACAP2 as Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6 supported histamine-evoked WPB exocytosis as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6

Direct observation of microtubule pushing by cortical dynein in living cells

ABSTRACT Microtubules are under the influence of forces mediated by cytoplasmic dynein motors associated with the cell cortex. If such microtubules are free to move, they are rap-idly transported inside cells. Here we directly observe fluorescent protein–labeled cortical dynein speckles and motile microtubules. We find that several dynein complex subunits, in-cluding the heavy chain, the intermediate chain, and the associated dynactin subunit Dctn1 (also known as p150glued) form spatially resolved, dynamic speckles at the cell cortex, which are preferentially associated with microtubules. Measurements of bleaching and dissociation kinetics at the cell cortex reveal that these speckles often contain multiple labeled dynein heavy-chain molecules and turn over rapidly within seconds. The dynamic behavior of micro-tubules, such as directional movement, bending, or rotation, is influenced by association with dynein speckles, suggesting a direct physical and functional interaction. Our results support a model in which rapid turnover of cell cortex–associated dynein complexes facilitates their search to efficiently capture and push microtubules directionally with leading plus ends