CryoET shows cofilactin filaments inside the microtubule lumen
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Abstract
Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
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Immunofluorescence staining for α-tubulin showed that the protrusions were filled with microtubules
Do you know if they also typically contain actin based on immunofluorescence or staining?
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actin filaments inside the microtubule lumen
Cofilactin in microtubule lumens?! What an amazing finding and paper! So much cool data here that I think will also lead to tons of really interesting hypotheses
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Cofilin is an actin binding protein
It might be nice to mention and discuss a little bit the link between Cytochalasin D and the actin cytoskeleton. Do you think that this could at all be affecting your results even with the low concentration? I think this could probably just be addressed in the text, but are there other methods to induce these protrusions that you could use as a control to show that this isn't related to Cytochalasin D or could you stain some cells with phalloidin and treat with this low concentration of CytD to show that it isn't affecting filaments?
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It is unclear how actin and cofilin get inside the microtubule.
This is a really interesting question! This is probably outside the scope of this paper, but related to this question, I'm curious about the timescale of formation of the protrusions? How dynamic are they?
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CytD
Does CtyD also decreases this phosphorylation a little? I'm wondering if the few luminal filaments in non-TG treated cells are also likely cofilactin as opposed to bare actin.
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13 protofilaments whereas minor fractions had 12 (2.8%), 14 (3.6%) or 15 (0.3%) protofilaments
Does the number of protofilaments change throughout the length of the microtubule? Like one end having more or less protofilaments or are they uniform throughout?
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