Good things still happen: a couple of weeks ago Nelson Leça successfully defended his PhD! 🥳 He did a fantastic job discussing his work and "wrestling" the exciting examination led by @jakedel.bsky.social and @pseudoenzyme.bsky.social.
It was a good day! 👏🎉

Fantastic seminar by Jake @jakedel.bsky.social on the wonders of the NDC80 complex, which never ceases to amaze.

Great seminar by Viji @viji-draviam.bsky.social at @i3suporto.bsky.social and very insightful discussions. Always a pleasure to host colleagues and brainstorm!

LEMOLish 🔬🥰

(13/16) Overall, our findings uncovered a mechanism where kinetochore-localized MPS1 indirectly contributes to pole-based error correction by proximity-driven activation of AAK at centrosomes! Further investigation into the structural basis of AAK activation by MPS1 is warranted (next on our list!).

(12/16) Inhibiting MPS1, even when KTs were close to centrosomes (CENP-Ei), reduced AAK activation and HEC1-S69 phosphorylation, supporting the conclusion that KT proximity-based activation of AAK by MPS1 potentiates phosphorylation of the bona fide error correction substrate HEC1 in human cells.

(11/16) Consistent with the extent of AAK activation, phosphorylation of HEC1 N-terminal tail (HEC1-S69Ph) followed the same trend – higher in CENP-Ei, lower in siSpindly cells.

(10/16) Consistent with there being spatial crosstalk between kinetochores and centrosomes in RPE-1 cells, AAK activation (AAK-T288Ph) at centrosomes/poles increased when kinetochores were closer (CENP-Ei cells) and decreased when they were further away (siSpindly cells)!

(9/16) We then teamed up with @barisiclab.bsky.social to see if proximity-based AAK activation by MPS1 is conserved. For that, we monitored AAK activation under experimental conditions where the positioning of kinetochores relative to centrosomes/spindle poles could be manipulated in RPE-1 cells.

(8/16) Consistent with the importance of AAK T360 phosphorylation for AAK activity, expressing the AAK-T360A mutant in S2 cells led to:
- reduced phosphorylation of the NDC80 tail (NDC80-S67Ph)
- failure to correct faulty kinetochore attachments
- delayed chromosome congression

(7/16) Importantly, AAK activation was compromised in cells depleted of NDC80 (which recruits MPS1 to kinetochores) or in cells expressing a non-phosphorylatable AAK-T360A mutant. This supports that kinetochore-recruited MPS1 drives AAK activation at centrosomes through T360 phosphorylation!

(6/16) Accordingly, the closer kinetochores were to centrosomes, the higher the AAK activation at centrosomes (measured by T-loop phosphorylation and a FRET sensor). Kinetochore proximity matters for AAK activation!

(5/16) Yes! AAK T360 phosphorylation does occur in cellulo and is needed for robust AAK activation at centrosomes! We generated an antibody against phospho-T360 that beautifully decorated centrosomes in an MPS1-dependent manner whenever chromosomes were in close proximity.

(4/16) In vitro, MPS1 directly phosphorylates a conserved threonine (T360) at AAK's C-lobe, significantly boosting AAK's own activation and AAK’s capacity to phosphorylate the N-terminal tail of HEC1 (a key AAK target for error correction). But does this happen in cells?

(3/16) However, the rescue by MPS1-CM2 was AURORA A (AAK)-dependent. Co-depleting AAK hindered the rescue, suggesting AAK was the one doing the heavy lifting (at least a good part of it). So, how does MPS1 talk to AAK?

(2/16) BUT then things got interesting! Nelson found that we could RESCUE these mitotic defects by simply tethering MPS1 to the centrosomes (MPS1-CM2). No MPS1 at kinetochores, only at centrosomes, and the error correction seems to work just fine!

(1/16) Nelson Leça, a PhD student in the lab, was exploring MPS1 roles across subcellular locations. He saw that depleting MPS1 in Drosophila cells increased the frequency of chromosomes erroneously attached to spindle microtubules and delayed chromosome congression. Nothing really new here...

Beautiful seminar by @dfachinetti.bsky.social