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Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin--rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling--induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor [alpha]--converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this 'sheddase' in regulating an actin-based structure.

The effects of the mutation, R416W, on glial fibrillary acidic protein (GFAP) assembly are described and R416W GFAP mutant is used to identify the early events in the development of Alexander disease, which is a rare and fatal neurodegenerative disorder. The monoclonal bodies specific for R416W GFAP have revealed the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers and the effects of the R416W GFAP are found to be dominant.

Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPK[alpha]1/[alpha]2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPK[alpha]2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPK[alpha]22 at the site of LKB1 phosphorylation ([Thr.sup.172]) or phosphorylation of acetylCoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB 1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1- expressing muscle. In contrast to AMPK[alpha]2. significant basal activity of AMPK[alpha]l was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPK[alpha]1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPK[alpha]2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPK[alpha]l in cardiac muscle. cellular energy metabolism; hypoxia: cardiovascular physiology; AMP-activated protein kinase

Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating [beta]-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Nathke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165-179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505-517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Nathke. 2001. Curr. Biol. 11:44-49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433-438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Nathke. 2001. Nat. Cell Biol. 3:429-432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Nathke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491-502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20: 5929-5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC.

Distinct endosomal vesicle populations arise due to specific endocytic pathways in growth factor-stimulated A431 cells. Coupling between macropinosomes and regular endosomes is not observed, but a definite differential sensitivity to the effects of brefeldin A of macropinosomes and endosomes is evident in the same cells. Macropinosomes joined with each other and recycle their contents to the cell surface.

Twenty Minosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other beta-rhizobial strains are examined by amplified rRNA gene restriction analysis. The 16S rRNA gene sequences of 15 of the 20 strains are determined and all are revealed to belong to the genus Burkholderia.

Explanation of the complex phenotype of the GPIdeAc null mutant, a model for the pathway of GPI biosynthesis in Trypanosoma brucei is presented. A model is presented which gives the pathway of GPI biosynthesis in T. brucei.