A rice class-XIV kinesin enters the nucleus in response to cold.

Higher plants possess a large number of kinesins, but lack the minus-end directed dynein motors. However, the kinesin class XIV has strongly expanded, and minus-end directed motors from this class may have taken over functions of cytoplasmic dyneins. In this study, we address the functional aspects of a novel rice homologue of the Arabidopsis class-XIV kinesins ATK1 and ATK5. Since a loss-of-function rice mutant of this kinesin is not viable, the function was studied in tobacco BY-2 as heterologous system. OsDLK-GFP stably expressed in BY-2 cells decorates cortical microtubules, but also can shift into the nucleus of interphase cells. Because of this peculiar localisation, we coined the name Dual Localisation Kinesin (DLK). The nuclear import of this protein is strongly and reversibly promoted in response to cold. During mitosis, OsDLK is repartitioned between spindle and phragmoplast. Motility assays in vitro using show that OsDLK can convey mutual sliding of microtubules and moves at a velocity comparable to other class-XIV kinesins. When tobacco cells overexpressing OsDLK are synchronised, they exhibit a delayed entry into metaphase, while the later phases of mitosis are accelerated. The data are discussed in relation to additional functions of this kinesin type, beyond their transport along microtubules.

The non-processive rice kinesin-14 OsKCH1 transports actin filaments along microtubules with two distinct velocities.

Microtubules and actin filaments function coordinately in many cellular processes(1-3). Although much of this coordination is mediated by proteins that statically bridge the two cytoskeletal networks(4-6), kinesin-14 motors with an actin binding calponin homology domain (KCHs) have been discovered as putatively dynamic crosslinkers in plants(7,8). OsKCH1, a KCH from rice, interacts with both microtubules and actin filaments in vivo and in vitro(9). However, it has remained unclear whether this interaction is dynamic or if actin binding reduces or even abolishes the motor's motility on microtubules(10,11). Here, we directly show in vitro that OsKCH1 is a non-processive, minus-end-directed motor that transports actin filaments along microtubules. Interestingly, we observe two distinct transport velocities dependent on the relative orientation of the actin filaments with respect to the microtubules. In addition, torsional compliance measurements on individual molecules reveal low flexibility in OsKCH1. We suggest that the orientation-dependent transport velocities emerge from OsKCH1's low torsional compliance combined with an inherently oriented binding to the actin filament. Together, our results imply a central role of OsKCH1 in the polar orientation of actin filaments along microtubules, and thus a contribution to the organization of the cytoskeletal architecture.

Initial boost release of transforming growth factor-ß3 and chondrogenesis by freeze-dried bioactive polymer scaffolds.

In cartilage regeneration, bio-activated implants are used in stem and progenitor cell-based microfracture cartilage repair procedures. Our aim was to analyze the chondrogenic potential of freeze-dried resorbable polymer-based polyglycolic acid (PGA) scaffolds bio-activated with transforming growth factor-ß3 (TGFB3) on human subchondral mesenchymal progenitor cells known from microfracture. Progenitor cells derived from femur heads were cultured in the presence of freeze-dried TGFB3 in high-density pellet culture and in freeze-dried TGFB3-PGA scaffolds for chondrogenic differentiation. Progenitor cell cultures in PGA scaffolds as well as pellet cultures with and without continuous application of TGFB3 served as controls. Release studies showed that freeze-dried TGFB3-PGA scaffolds facilitate a rapid, initial boost-like release of 71.5% of TGFB3 in the first 10 h. Gene expression analysis and histology showed induction of typical chondrogenic markers like type II collagen and formation of cartilaginous tissue in TGFB3-PGA scaffolds seeded with subchondral progenitor cells and in pellet cultures stimulated with freeze-dried TGFB3. Chondrogenic differentiation in freeze-dried TGFB3-PGA scaffolds was comparable to cultures receiving TGFB3 continuously, while non-stimulated controls did not show chondrogenesis during prolonged culture for 14 days. These results suggest that bio-activated, freeze-dried TGFB3-PGA scaffolds have chondrogenic potential and are a promising tool for stem cell-mediated cartilage regeneration.