Interactions between two regulatory proteins of microtubule dynamics, HDAC6, TPPP/p25, and the hub protein, DYNLL/LC8.

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6. Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network.

T-complex-associated-testis-expressed 3 (TCTE3) is a novel marker for pancreatobiliary carcinomas.

Several markers of pancreatobiliary lineage have been described in the literature. However, none have demonstrated sufficient specificity and sensitivity to warrant diagnostic use. We evaluated the utility of T-complex-associated-testis-expressed 3 (TCTE3) as a pancreatobiliary marker. A set of 247 adenocarcinomas from the gastrointestinal (GI) tract was identified including 18 from the gastroesophageal junction (GEJ), 29 stomach, 17 ampullary, 62 pancreatic, and 16 common bile duct and gallbladder (CBD/GB), 13 non-ampullary small intestine, 32 colon, and 24 rectum. The remainder consisted of 16 cholangiocarcinomas and 20 hepatocellular carcinomas (HCC). Additionally, 163 adenocarcinomas from the breast, gynecologic tract, prostate, urothelium, kidney, and lung were stained for comparison. Immunohistochemistry for TCTE3 and other gastrointestinal markers was performed. Positive expression of TCTE3 was characterized by a strong, well-defined membranous pattern with or without weak cytoplasmic staining. Expression was identified in the normal epithelial cells of pancreatobiliary tree, but staining was absent in normal epithelial cells of esophagus, stomach, and intestine. Hepatocytes, pancreatic acini and islets and other non-epithelial cells were also negative for staining. TCTE3 was expressed in 93.5% of pancreatic ductal adenocarcinomas, 37.5% of CBD/GB adenocarcinomas, 50% of cholangiocarcinomas, 76.4% of ampullary adenocarcinomas, and 33.3% of GEJ adenocarcinomas. Only 3.5% of the gastric, 7.7% of non-ampullary small intestinal and 6.25% of colonic tumors exhibited positive staining. Expression was absent in rectal carcinomas and HCCs. These results suggest that TCTE3 is a useful marker of pancreatobiliary differentiation and may aid in distinguishing these tumors from gastric and intestinal primary tumors.

Dynein motion switches from diffusive to directed upon cortical anchoring.

Cytoplasmic dynein is a motor protein that exerts force on microtubules. To generate force for the movement of large organelles, dynein needs to be anchored, with the anchoring sites being typically located at the cell cortex. However, the mechanism by which dyneins target sites where they can generate large collective forces is unknown. Here, we directly observe single dyneins during meiotic nuclear oscillations in fission yeast and identify the steps of the dynein binding process: from the cytoplasm to the microtubule and from the microtubule to cortical anchors. We observed that dyneins on the microtubule move either in a diffusive or directed manner, with the switch from diffusion to directed movement occurring upon binding of dynein to cortical anchors. This dual behavior of dynein on the microtubule, together with the two steps of binding, enables dyneins to self-organize into a spatial pattern needed for them to generate large collective forces.

Dynein LIC1 localizes to the mitotic spindle and midbody and LIC2 localizes to spindle poles during cell division.

CD-1 (cytoplasmic dynein-1) is a multisubunit motor protein complex involved in intracellular trafficking and mitosis. The dynein LIC (light intermediate chain) subunits, LIC1 (DLIC-1, gene symbol DYNC1LI1) and LIC2 (DLIC-2, gene symbol DYNC1LI2), associate with the dynein HC (heavy chain) in a mutually exclusive manner and thus define distinct functional CD-1 complexes. Here, we analysed the mitotic distribution of LIC1 and LIC2. We found that from metaphase through anaphase, LIC1 localizes to the mitotic spindle and concentrates within the midbody during the abscission step of cytokinesis. Conversely, LIC2 strongly localizes to the spindle poles from prophase through telophase. These data suggest distinct functions for LIC1 and LIC2-containing CD-1 complexes during cell division.