Activating mutations in the Kras gene are commonly found in some but not all epithelial cancers. In order to understand the susceptibility of different epithelial tissues to Kras-induced tumorigenesis, we introduced one of the most common Kras mutations, KrasG12D, broadly in epithelial tissues. We used a mouse model in which the G12D mutation is placed in the endogenous Kras locus controlled by inducible, Cre-mediated recombination in tissues expressing cytokeratin 19 including the oral cavity, GI tract, lungs, and ducts of the liver, kidney, and the pancreas. Introduction of the KrasG12D mutation in adult mouse tissues led to neoplastic changes in some but not all of these tissues. Notably, many hyperplasias, metaplasias and adenomas were observed in the oral cavity, stomach, colon and lungs, suggesting that exposure to products of the outside environment promotes KrasG12D-initiated tumorigenesis. However, environmental exposure did not consistently correlate with tumor formation, such as in the small intestine, suggesting that there are also intrinsic differences in susceptibility to Kras activation. The pancreas developed small numbers of mucinous metaplasias with characteristics of early stage pancreatic intraepithelial neoplasms (PanINs), supporting the hypothesis that pancreatic ducts have the potential to give rise pancreatic cancer.

Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single “canonical” site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate (“noncanonical”) sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal.

Saccharomyces cerevisiae kinesin-5 Cin8 displays unconventional biochemical behavior including bidirectional motility and ability to bind multiple motor domains per αβ tubulin dimer in the microtubule lattice. Previous research suggested that a large loop-8 insert near the microtubule binding interface of Cin8 was critical for its noncanonical microtubule binding behavior. Here we utilized mutagenesis, thermodynamic, and kinetic assays to further understand the mechanism for how this loop-8 insert promotes super-stoichiometric microtubule binding in Cin8. This loop-8 insert that interrupts the conserved β5a/b hairpin was swapped between Cin8, Eg5 (KIF11, a human kinesin-5) and Kip1 (another S. cerevisiae kinesin-5). Cin8 with the loop-8 insert from Eg5 (Cin8-EL8) binds one motor per tubulin dimer, whereas Eg5 with the loop-8 insert from Cin8 (Eg5-CL8) binds approximately 2-4 motors per tubulin dimer. Eg5-CL8 bound the canonical and noncanonical sites on the microtubule lattice with weakened oligomerization between motors, while Cin8-EL8 showed only canonical site binding. These results demonstrate that the large loop-8 insert in Cin8 is necessary and sufficient to promote noncanonical microtubule binding behavior.

The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclindependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear.Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to-but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its directionality from fast minus-to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end directionality.

The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclindependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear.Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to-but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its directionality from fast minus-to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end directionality.