The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.

Summary Na + /K + -ATPase, an integral membrane protein, has been studied for over a half century with respect to its transporter function in the plasma membrane, where it expels three Na + ions from the cell in exchange for two K + ions. In this study, we demonstrate a functioning Na + /K + -ATPase within HEK293 cell nuclei. This subcellular localization was confirmed by western blotting, ouabain-sensitive ATPase activity of the nuclear membrane fraction, immunocytochemistry and delivery of fluorescently tagged Na + /K + -ATPase a-and bsubunits. In addition, we observed an overlap between nuclear Na + /K + -ATPase and Na/Ca-exchanger (NCX) when nuclei were immunostained with commercially available Na + /K + -ATPase and NCX antibodies, suggesting a concerted physiological coupling between these transporters. In keeping with this, we observed an ATP-dependent, strophanthidin-sensitive Na + flux into the nuclear envelope (NE) lumen loaded with the Na-sensitive dye, CoroNa-Green. Analogous experiments using Fluo-5N, a low affinity Ca 2+ indicator, demonstrated a similar ATP-dependent and strophanthidin-sensitive Ca 2+ flux into the NE lumen. Our results reveal an intracellular physiological role for the coordinated efforts of the Na + /K + -ATPase and NCX to actively remove Ca 2+ from the nucleoplasm into the NE lumen (i.e. the nucleoplasmic reticulum).

Microtubule associated proteins (MAPs) are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band, and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification, transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis. This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment. R (2005). A minus-enddirected kinesin with plus-end tracking protein activity is involved in spindle morphogenesis. Mol. Biol. Cell 16, 1584-1592. Amos LA, Schlieper D (2005). Microtubules and maps. Adv. Protein Chem. 71, 257-298. Baluska F, Menzel D, Barlow PW (2006). Cytokinesis in plant and animal cells: Endosomes 'shut the door'. Dev. Biol. 294, 1-10. Bichet A, Desnos T, Turner S, Grandjean O, Hofte H (2001). BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis. Plant J. 25, 137-148. Binarova P, Cenklova V, Prochazkova J, Doskocilova A, Volc J, Vrlik M et al. (2006). Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis. Plant Cell 18, 1199-1212. Bisgrove SR, Hable WE, Kropf DL (2004). +TIPs and microtubule regulation. The beginning of the plus end in plants. Plant Physiol. 136, 3855-3863. Bouquin T, Mattsson O, Naested H, Foster R, Mundy J (2003). The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth. J. Cell Sci. 116, 791-801. Burk DH, Liu B, Zhong RQ, Morrison WH, Ye ZH (2001). A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell 13, 807-827. Burk DH, Ye ZH (2002). Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein. CW (1999). The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. Proc. Natl. Acad. Sci. USA 96, 14931-14936. Chan J, Calder GM, Doonan JH, Lloyd CW (2003a). EB1 reveals mobile microtubule nucleation sites in Arabidopsis. Nat. Cell Biol. 5, 967-971. Chan J, Mao GJ, Smertenko A, Hussey PJ, Naldrett M, Bottrill A et al.(2003b). Identification of a MAP65 isoform involved in directional expansion of plant cells. FEBS Lett. 534, 161-163. Chan J, Calder G, Fox S, Lloyd C (2007). Cortical microtubule arrays undergo rotary movements in Arabidopsis hypocotyl epidermal cells. Nat. Cell Biol. 9, 171-175. Chang HY, Smertenko AP, ...

Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3–C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis.

Blocking efficacy of whole soymilk, non-fat soymilk, SuperBlock™, and non-fat milk was evaluated by performing standard protein immunoblotting procedures on both purified protein and crude nuclear extracts from HEK 293 cells. Non-fat soymilk was found to have superior blocking efficacy compared to other blocking agents in terms of high signal to noise ratio with the shortest blocking times. In addition, the presence of low concentrations of the detergent Tween-20 (0.05–0.1% v/v) in the wash buffer, as well as, antibody incubations significantly lessened the background compared to only including the detergent during wash steps.