It was fantastic to work w. Ved & Rhiju! Ved was incredibly productive & received a R21 for this work.

This was a project that survived COVID, 2 lab moves, sabbatical...

We're fortunate & grateful to Brad Zuchero’s lab for help w. oligo cultures after I left Stanford 🙏

8/8

Combined w. work from Jackie Trotter & Doug Fields Labs, we propose a RNA switch model:

- During transport, hnRNP-F binds unstructured MLS to inhibit translation.

- At the cell periphery, membrane-bound Fyn kinase phosphorylates & releases hnRNP-F to initiate translation.

7/8

Next, we used mass spec to ask what proteins associate with the MLS mRNA region.

We found hnRNP-F and proteins involved in translation!

Our structure of the MLS also had the canonical hnRNP-F-binding GGG(A) motifs!

6/8

Importantly, the MLS can be used to deliver CDS cargos throughout oligodendrocyte processes.

Here, we used EGFP as a reporter cargo.

5/8

We found a ~120-nt region sufficient for transport that we call the MLS.

Comparing to decades-old results from the Carson lab:
- Their RTS regions was not validated.
- But our MLS partially overlaps with their RLR region.

4/8

We asked which part of the ~1.5-kb Mbp 3' UTR is important for mRNA transport?

Ved applied 2 orthogonal techniques to primary olives:
- DMS-MaPseq to solve the RNA structure
- SLAP-seq - a method he innovated combining ~270 reporters and Boyden chambers cultures

3/8

Mbp (myelin basic protein) is the most abundant mRNA in oligodendrocytes

MBP functions in compaction, a bizarre cellular phenomenon that extrudes cytoplasm from the myelin sheath. It acts as a molecular glue between adjacent membranes. Hence, it is locally translated.

2/8

That’s terrifying 😞… so many US scientists in our field have DOD (Dept. of Defense) grants, but it doesn’t mean they’re working on anything that harms people (usually working on diseases that affect veterans) and certainly they’ve never been targeted like this.

Great first post!

We have a pink frog in our lab today… already knocked over an Erlenmeyer 😆

Don’t forget rejecting submissions because it’s over the word count… before it even gets seen by a scientist! Dealing with that now 😞

Thanks Kathrin! Meghan (first co-author) did the actin imaging that you saw and of course you know Lana 🤓

Thanks! Reposted link.

So this preprint opens up many new questions in astrocyte cell biology!

This was a real team effort:
- Postdoc Will Barclay started the project at the NIH
- Postbac Johanna Bergstrom and undergrad Eva Lopez analyzed data
- PhD student Lana Ho contributed imaging data

Many distal ends are enriched in GFAP.

We don't know what this means yet - maybe these can become endfeet that contact blood vessels. Astrocyte processes are heterogeneous in vivo (some contact blood vessels, others contact synapses) - like axons vs. dendrites in neurons.

7/x

By cryo-ET, we could see a lot of intermediate filaments (IFs). The ratio of IFs:microtubules was very high ~7:1.

Image below shows: IFs (blue), microtubules (green), actin (yellow).

6/x

Microtubules in primary processes are highly stable as indicated by:
- Acetylation
- Detyrosination
- MIPs (microtubule inner proteins)
- IF content

5/x