Titin (Visco-) Elasticity in Skeletal Muscle Myofibrils

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any unfolding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin’s visco-elastic properties appear to depend on the Ig domain un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.     

Microtubule assembly dynamics: new insights at the nanoscale

Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.     

Effects of plant height and row spacing on kenaf forage potential with multiple harvests

Kenaf (Hibiscus cannabinus L.) forage potential can be enhanced through its regrowth capacity and higher production in narrow rows. A field experiment was conducted in Matamoros, Coahuila, Mexico, during 2 growing seasons (2004 and 2005) to study the effects of plant height and row spacing on kenaf forage potential with multiple harvests. This study evaluated the effects of (1) 2 plant heights at cutting (1.0-1.2 m and 1.8-2.0 m) and (2) 4 inter row spacings (0.19, 0.38, 0.57 and 0.76 m) using a 2 x 4 factorial arrangement of treatments in a completely randomized block design with 4 replications. Dry matter (DM) and crude protein (CP) yields, DM partitioning, neutral detergent fiber (NDF) and CP concentrations were determined. Heights at cutting × row spacing interactions were not significant for the monitored variables (p>0.05). Kenaf response to treatments was only relevant for main effects (p≤0.05). Row spacing and plant height affected DM and CP yields (p≤0.05), whereas only plant height affected chemical composition and DM partitioning (p≤0.05). Dry matter (17.0%-26.0%), and CP (12.4%-15.6%) yields were higher (p≤0.05) when plant heights had reached 1.8 to 2.0 m. Row spacing reduction from 0.76 m to 0.38 and 0.19 m increased DM yield (20.4-33.4%) and CP yield (24.2-38.5%) (p≤0.05). Kenaf forage potential increases when planted in narrow rows and harvested 2 or 3 times during the growing season.     

Anatomical and chemical characteristics of the seed coat of <i>Medicago sativa</i> L. (alfalfa) cv. Baralfa 85 seeds and their association with seed dormancy

Seeds of alfalfa (Medicago sativa L.) can exhibit seedcoat imposed dormancy, which produces hard seeds within a seed lot. These seeds do not germinate because they do not imbibe water due to a barrier to water entry in the seed coat. The aim of this work was to analyze the anatomical and chemical characteristics of the testa of alfalfa seeds with respect to water permeability levels. The anatomy of seeds of the cv. Baralfa 85 was studied and structural substances, polyphenols, tannins and cutin present in the testa of seeds of different water permeability levels were determined. The anatomical characteristics of the seed coat and the proportions of components were found to determine the permeability level of the seed coat, an aspect that is associated with the physical seed dormancy level. Anatomically, increased thickness of the testa was associated with a lower permeability level. The difference may be attributed to the variation in cuticle thickness, length of macrosclereids and thickness of the cell wall, and presence and development of osteosclereids. From the physiological and chemical points of view, the mechanism of physical dormancy of the testa is explained by a greater amount of components that repel water and cement the cell wall, such as polyphenols, lignins, condensed tannins, pectic substances, and a lower proportion of cellulose and hemicellulose.     

Interactions between CLIP-170, tubulin, and microtubules: implications for the mechanism of Clip-170 plus-end tracking behavior

CLIP-170 belongs to a group of proteins (+TIPs) with the enigmatic ability to dynamically track growing microtubule plus-ends. CLIP-170 regulates microtubule dynamics in vivo and has been implicated in cargo-microtubule interactions in vivo and in vitro. Though plus-end tracking likely has intimate connections to +TIP function, little is known about the mechanism(s) by which this dynamic localization is achieved. Using a combination of biochemistry and live cell imaging, we provide evidence that CLIP-170 tracks microtubule plus-ends by a preassociation, copolymerization, and regulated release mechanism. As part of this analysis, we find that CLIP-170 has a stronger affinity for tubulin dimer than for polymer, and that CLIP-170 can distinguish between GTP- and GDP-like polymer. This work extends the previous analysis of CLIP-170 behavior in vivo and complements the existing fluorescence microscope characterization of CLIP-170 interactions with microtubules in vitro. In particular, these data explain observations that CLIP-170 localizes to newly polymerized microtubules in vitro but cannot track microtubule plus-ends in vitro. These observations have implications for the functions of CLIP-170 in regulating microtubule dynamics.     

Geographical patterns of genetic variation in the world collections of wild annual Cicer characterized by amplified fragment length polymorphisms

Cicer reticulatum, C. echinospermum, C. bijugum, C. judaicum, C. pinnatifidum, C. cuneatum and C. yamashitae are wild annual Cicer species and potential donors of valuable traits to improve chickpea (C. arietinum). As part of a large project to characterize and evaluate wild annual Cicer collections held in the world gene banks, AFLP markers were used to study genetic variation in these species. The main aim of this study was to characterize geographical patterns of genetic variation in wild annual Cicer germplasm. Phylogenetic analysis of 146 wild annual Cicer accessions (including two accessions in the perennial C. anatolicum and six cultivars of chickpea) revealed four distinct groups corresponding well to primary, secondary and tertiary gene pools of chickpea. Some possible misidentified or mislabelled accessions were identified, and ILWC 242 is proposed as a hybrid between C. reticulatum and C. echinospermum. The extent of genetic diversity varied considerably and was unbalanced between species with greatest genetic diversity found in C. judaicum. For the first time geographic patterns of genetic variation in C. reticulatum, C. echinospermum, C. bijugum, C. judaicum and C. pinnatifidum were established using AFLP markers. Based on the current collections the maximum genetic diversity of C. reticulatum, C. echinospermum, C. bijugum and C. pinnatifidum was found in southeastern Turkey, while Palestine was the centre of maximum genetic variation for C. judaicum. This information provides a solid basis for the design of future collections and in situ conservation programs for wild annual Cicer.     

p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B

p21-activated kinase 1 (Pak1) induces cytoskeleton reorganization in part by regulating microtubule dynamics through an elusive mechanism. Using a yeast two-hybrid screen, we identified tubulin cofactor B (TCoB) (a cofactor in the assembly of the alpha/beta-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of gamma-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics.     

Random peptide bacteriophage display as a probe for urokinase receptor ligands

The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.     

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.