She1-Mediated Inhibition of Dynein Motility along Astral Microtubules Promotes Polarized Spindle Movements

Abstract

SUMMARY Background Cytoplasmic dynein motility along microtubules is critical for diverse cellular processes ranging from vesicular transport to nuclear envelope breakdown to mitotic spindle alignment. In yeast, we have proposed a regulated-offloading model to explain how dynein motility drives microtubule sliding along the cortex, powering transport of the nucleus into the mother-bud neck [1, 2]: the dynein regulator She1 limits dynein offloading by gating the recruitment of dynactin to the astral microtubule plus end, a prerequisite for offloading to the cortex. However, whether She1 subsequently affects cortically anchored dynein activity during microtubule sliding is unclear. Results Using single-molecule motility assays, we show that She1 strongly inhibits dynein movement along microtubules, acting directly on the motor domain in a manner independent of dynactin. She1 has no effect on the motility of either Kip2, a kinesin that utilizes the same microtubule track as dynein, or human kinesin-1, demonstrating the specificity of She1 for the dynein motor. At single-molecule resolution, She1 binds tightly to and exhibits diffusional behavior along microtubules. Diffusive She1 collides with and pauses motile dynein motors, prolonging their attachment to the microtubule. Furthermore, Aurora B/Ipl1 directly phosphorylates She1 and this modification appears to enhance the diffusive behavior of She1 along microtubules and its potency against dynein. In cells, She1 dampens productive microtubule-cortex interactions specifically in the mother compartment, polarizing spindle movements toward the bud cell. Conclusions Our data reveal how inhibitory microtubule-associated proteins selectively regulate motor activity to achieve unidirectional nuclear transport, and demonstrate a direct link between cell cycle machinery and dynein pathway activity.

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

“…Construction of Plasmids-For bacterial expression, desired nucleotide sequences of human TPX2 constructs (full-length or truncated at amino acid 710) were cloned into a pGEX vector following an N-terminal GST tag and a ULP1 protease cleavage site (15). At the C terminus of TPX2, the stop codon was removed, and the Halo tag sequence was introduced.…”

Regulation of Mitotic Spindle Disassembly by an Environmental Stress-Sensing Pathway in Budding Yeast

“…Although She1 still localizes to the bud neck in sho1D, pbs2D, and hog1D cells, it strongly decorates the spindle at inappropriate times during anaphase, as if it cannot be excluded or removed from the spindle midzone. This finding highlights the somewhat paradoxical nature of She1: although She1 binds and diffuses along microtubules in vitro (Markus et al 2012) and is required for timely spindle disassembly in wild-type cells ; this study), it does not strongly localize to the site most critical to the destabilization of spindle microtubules during anaphase, the midzone. Moreover, She1 localization in the HOG pathway mutants and boi2D cells shows that enriching She1 at the midzone, or in the case of the sho1D mutant even at the depolymerizing ends of spindle microtubules, does not promote spindle disassembly.…”

“…Together, these three classes of MTs form the mitotic spindle. Over the course of mitosis, the dynamics of microtubules are modulated by several classes of proteins including +TIP proteins such as EB1/Bim1, which stabilize microtubule plus ends and promote growth, crosslinking proteins such as Ase1, which stabilize lateral interactions between ipMTs of the midzone, motor proteins such as Cin8 and Kip1, which generate forces necessary to slide the ipMTs by each other, and other motor proteins such as Kar3 and dynein (reviewed in Westermann et al 2007;Khmelinskii and Schiebel 2008) that are also responsible for generating forces on MTs.She1 is an important regulator of microtubule dynamics in budding yeast and it appears to have several functions in the cell Bergman et al 2012;Markus et al 2012). Beginning in G1, She1 localizes to the SPB and restricts the activity of dynein by inhibiting its interaction with dynactin until anaphase, when dynein is required for spindle positioning prior to elongation into the bud ).…”

“…She1 is an important regulator of microtubule dynamics in budding yeast and it appears to have several functions in the cell Bergman et al 2012;Markus et al 2012). Beginning in G1, She1 localizes to the SPB and restricts the activity of dynein by inhibiting its interaction with dynactin until anaphase, when dynein is required for spindle positioning prior to elongation into the bud ).…”

Mechanism of Nuclear Movements in a Multinucleated Cell

“…The pulling of a cMT by "walking" of cortex-anchored dynein toward the cMT minus end at the SPB does not trigger depolymerization of this cMT, and the cMT slides head on with its plus end along the cortex. In addition, in contrast to higher eukaryotes, directing nuclei by dynein-mediated cortical pulling is restricted in S. cerevisiae to a small window of the cell cycle regulated by the inhibitor She1 (Woodruff et al, 2009;Markus et al, 2012).…”