She1 affects dynein through direct interactions with the microtubule and the dynein microtubule-binding domain

Abstract

Cytoplasmic dynein is an enormous minus end-directed microtubule motor. Rather than existing as bare tracks, microtubules are bound by numerous microtubule-associated proteins (MAPs) that have the capacity to affect various cellular functions, including motor-mediated transport. One such MAP is She1, a dynein effector that polarizes dynein-mediated spindle movements in budding yeast. Here, we characterize the molecular basis by which She1 affects dynein, providing the first such insight into which a MAP can modulate motor motility. We find that She1 affects the ATPase rate, microtubule-binding affinity, and stepping behavior of dynein, and that microtubule binding by She1 is required for its effects on dynein motility. Moreover, we find that She1 directly contacts the microtubule-binding domain of dynein, and that their interaction is sensitive to the nucleotide-bound state of the motor. Our data support a model in which simultaneous interactions between the microtubule and dynein enables She1 to directly affect dynein motility.

IGF 1R regulates retrograde axonal transport of signalling endosomes in motor neurons

“…Given that IGF1R‐null mice have been found to have hyperphosphorylated tau , it is possible that acute modulation of IGF1R could alter the presence of tau on microtubules, thus influencing transport. Findings from recent work implicate MAPs further and demonstrate that the MAP she1 can directly influence the ATPase activity of dynein and reduce the stepping frequency of the motor . The precise role of MAPs in modulating retrograde axonal transport after IGF1R modulation should be investigated in the future.…”

Molecular basis for dyneinopathies reveals insight into dynein regulation and dysfunction

“…Moreover, by scoring for "neck transit" success frequency -events in which a dynein-dynactin-mediated spindle translocation results in successfully transiting the narrow mother-bud neck -we are also able to determine if there are potential defects in force production. Previous studies have shown that neck transits are compromised in cases where dynein's microtubulebinding affinity is weakened 70 , or when the CAP-gly domain of Nip100 (homolog of human p150 component of the dynactin complex) is genetically ablated 69 . Subsequent to HU arrest, full Z-stacks of the mitotic spindle and astral microtubules (via GFP-Tub1) were acquired every 10 seconds (see Fig.…”

Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

“…We wondered whether the apparent increase in dynein activity in tub1 G437R cells could be a consequence of reduced microtubule binding by the microtubule associated protein (MAP) She1, a dynein inhibitor (Markus et al, 2012;Woodruff et al, 2009). Since She1 inhibitory activity requires its microtubule binding activity (Ecklund et al, 2017), we first tested the effect of G437R on the interaction between She1 and tubulin. A She1-Gal4 activation domain (AD) fusion was tested for a two-hybrid interaction with either Tub1 or Tub1 G437R , the latter of which were fused to the LexA DNA-binding domain (LexADBD).…”

“…Like many MAPs, the interaction between She1 and microtubules requires the disordered C-terminal tails of tubulin (Ecklund et al, 2017;Markus et al, 2012). Thus, to further confirm the importance of G437 in the She1-tubulin interaction, we performed a pull-down assay in which this interaction was competitively inhibited by addition of a peptide encompassing the C-terminus of Tub1 (amino acids 415-447; both She1 and tubulin were used at 5 µg/ml or below, well below the critical concentration required for microtubule assembly).…”

“…The extent of dynein activity is largely governed by its localization to these sites; however, as in higher 5 eukaryotes (Tan et al, 2019), at least one known MAP can also regulate dynein activity in cells: She1. The precise mechanism by which it does so in cells is currently unclear; however, in vitro studies show that She1 can reduce dynein velocity through simultaneous interactions with both microtubules and dynein (Ecklund et al, 2017), whereas live cell studies have shown that She1 plays a role in polarizing dyneinmediated spindle movements toward the daughter cell (Markus et al, 2012), perhaps in part by tuning dynactin recruitment to plus end-associated dynein Woodruff et al, 2009).…”