The N-terminal TOG domain of Arabidopsis MOR1 modulates affinity for microtubule polymers.

Microtubule-associated proteins of the highly conserved XMAP215/Dis1 family promote both microtubule growth and shrinkage, and move with the dynamic microtubule ends. The plant homologue, MOR1, is predicted to form a long linear molecule with five N-terminal TOG domains. Within the first (TOG1) domain, the mor1-1 leucine to phenylalanine (L174F) substitution causes temperature-dependent disorganization of microtubule arrays and reduces microtubule growth and shrinkage rates. By expressing the two N-terminal TOG domains (TOG12) of MOR1, both in planta for analysis in living cells and in bacteria for in vitro microtubule-binding and polymerization assays, we determined that the N-terminal domain of MOR1 is crucial for microtubule polymer binding. Tagging TOG12 at the N-terminus interfered with its ability to bind microtubules when stably expressed in Arabidopsis or when transiently overexpressed in leek epidermal cells, and impeded polymerase activity in vitro. In contrast, TOG12 tagged at the C-terminus interacted with microtubules in vivo, rescued the temperature-sensitive mor1-1 phenotype, and promoted microtubule polymerization in vitro. TOG12 constructs containing the L174F mor1-1 point mutation caused microtubule disruption when transiently overexpressed in leek epidermis and increased the affinity of TOG12 for microtubules in vitro. This suggests that the mor1-1 mutant protein makes microtubules less dynamic by binding the microtubule lattice too strongly to support rapid plus-end tracking. We conclude from our results that a balanced microtubule affinity in the N-terminal TOG domain is crucial for the polymerase activity of MOR1.

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.

Return-to-play probabilities following new versus recurrent ankle sprains in high school athletes.

OBJECTIVES: Although ankle sprains have the highest recurrence rate of any musculoskeletal injury, objective estimates of when an athlete is likely to return-to-play (RTP) are unknown. The purpose was to compare time to return-to-play probability timelines for new and recurrent ankle sprains in interscholastic athletes. DESIGN: Observational. METHODS: Ankle sprain data were collected at seven high schools during the 2007-2008 and 2008-2009 academic years. Ankle sprains were categorized by time lost from participation (same day return, next-day return, 3-day return, 7-day return, 10-day return, >22-day return, no return [censored data]). Time-to-event analyses were used to determine the influence of ankle injury history on return-to-play after an ankle sprain. RESULTS: 204 ankle sprains occurred during 479,668 athlete-exposures, 163 were new (4 censored) and 35 recurrent (1 censored). There was no significant difference (p=0.89) between the time-to-event curves for new and recurrent ankle sprains. The median (inter-quartile rage) time to return-to-play for new sprains (inter-quartile range)=3 days (same day to 7 day return); recurrent sprains=next day return (next day to 7 day return). Noteworthy probabilities [95% CIs] include: same day return (new=25.2[18.7, 31.9], recurrent=17.1[6.6, 30.3]); next-day return (new=43.6[35.3, 52.7], recurrent=51.4[32.5, 67.5]); and 7-day return (new=85.9[73.8, 94.4], recurrent=94.3[47.8, 99.5]). CONCLUSIONS: Previous injury history did not affect time until return-to-play probabilities for ankle sprains. Time until return-to-play analyses that describe the likelihood of return-to-play are useful to clinicians by providing prognostic guidelines and can be used for educating athletes, coaches, and parents about the likely timeframe of being withheld from play.

MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root.

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.