Comparison of a novel extracapsular suture technique with a standard fabellotibial suture technique for cranial cruciate ligament repair using a custom-made limb-press model in cats.

OBJECTIVES: The aim of our study was to compare the standard fabellotibial suture with Mini TightRope fixation for the treatment of a cranial cruciate ligament (CCL) rupture using a feline custom-made limb press. METHODS: Cadaveric hindlimbs of 10 cats were inserted in the limb press at predefined joint angles and loads of 10% and 30% body weight (BW) were applied. Mediolateral radiographs were taken and three-dimensional coordinates were recorded using a microscribe digitiser, with intact and transected CCLs and after either fabellotibial suture or Mini TightRope fixation were performed. Different distances and angles from radiographs or microscribe coordinates were analysed. RESULTS: Radiographic distances from the femoral condyle to the cranial edge of the tibia (X1-X2) were higher in CCL-deficient stifles than in intact stifles at 10% and 30% BW loads. All fabellotibial sutures and Mini TightRope fixations neutralised excessive cranial tibial thrust. A significant difference in the distance between the patella and tibial tuberosity (D2) was observed between CCL-deficient limbs and Mini TightRope-fixed limbs at 10% BW load (P <0.04). A significant difference in the distance between the tibial tuberosity and lateral collateral ligament of the femur (D3) was observed between the intact and transected CCLs on the left legs at 10% BW load (P <0.003) and on both legs at 30% BW load (P <0.002). Furthermore, we observed significant differences between CCL-deficient left legs and Mini TightRope-treated legs at 10% BW load (P <0.003). With regard to fabellotibial suture-treated legs, we observed significant differences between transected limbs and fixed limbs at 30% BW load (P <0.004). W1 (craniocaudal angle) and W2 (mediolateral angle) showed significant differences between intact and transected CCLs and between transected and fixed limbs at 30% BW load (P <0.004). CONCLUSIONS AND RELEVANCE: Fixation of CCL-deficient stifles with lateral fabellotibial suture, as well as Mini TightRope tightened with a 20 N load, produces good biomechanical stability, as detected via radiographic assessment.

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 missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.

Alternating-site mechanism of kinesin-1 characterized by single-molecule FRET using fluorescent ATP analogues.

Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of approximately 1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way.