Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons
- Feinberg School of Medicine, Northwestern University, United States
Figures
Figure 1 with 1 supplement
Microtubule minus-ends push the plasma membrane during the initial stages of neurite outgrowth.
(A) Model of microtubule-microtubule sliding driven by kinesin-1. Kinesin-1 slides antiparallel microtubules apart with their minus-ends leading (left panel). When kinesin-1 binds to parallel …
Figure 1—figure supplement 1
CAMSAP3 labels minus-ends of microtubules in Drosophila S2 cells.
(A) Microtubules in S2 cells expressing GFP-CAMSAP3 were fragmented by 1 hr treatment with 25 µM vinblastine; cells were fixed and stained with primary antitubulin antibody DM1α and TRITC-labeled …
Figure 2 with 2 supplements
Dynein specifies microtubule orientation in axons.
(A and B) Axonal microtubules gradually acquire uniform orientation during development. (A) Representative still images of EB1-GFP expressing neurons cultured for 4 hr, 21 hr, or 48 hr. Kymographs …
Figure 2—figure supplement 1
Knockdown efficiency of DHC and dynein cofactors in Drosophila S2 cells.
(A) Western blot analysis of S2 cell extracts obtained from control (untreated) or DHC dsRNA-treated cells using anti-DHC and anti-KHC (loading control) antibodies. (B) Temporal color code …
Figure 2—figure supplement 2
Distribution of GFP-CAMSAP3 in DHC RNAi S2 processes.
(A) Distribution of GFP-CAMSAP3 in DHC depleted S2 process. CAMSAP3 molecules accumulate mainly at the end of the tip of the processes (upper panel). Adjustment of contrast in the boxed area reveals …
Figure 3 with 1 supplement
Dynein recruitment to the cortex is required for microtubule sorting.
(A) Actin depolymerization in cultured neurons results in formation of axons with antiparallel microtubules. Graph depicts the fraction of axonal EB1 comets moving in each direction in 48 hr-cultured…
Figure 3—figure supplement 1
Validation of the rapalog recruitment assays in S2 cells.
(A) S2 cells expressing FRB-GFP and PEX3-RFP-FKBP before (upper panels) and after (bottom panels) addition of rapalog. Note that in the presence of rapalog, GFP signal concentrates to peroxisomes. …
Figure 4
Model of microtubule sliding and axon formation.
(A) Kinesin-1 induced sliding of antiparallel microtubules initiates formation of processes. Note that at this stage cortical dynein can only slide microtubules parallel to the plasma membrane, …
Videos
Video 1
Microtubules slide in both directions in Drosophila-cultured neurons.
Time-lapse video of photoconverted microtubules in Drosophila-cultured neurons expressing tdEOS-αtubulin. A small area of the nascent axon was photoconverted by 405 nm light. Note that microtubules …
Video 2
CAMSAP3 decorates microtubule-ends in Drosophila cells.
Related to Figure 1B. A time-lapse video of S2 cells expressing GFP-CAMSAP3 and mCherry-tubulin. Scale bar 10 µm.
Video 3
Microtubule plus-ends and minus-ends binding proteins do not colocalize in Drosophila S2 cells.
Related to Figure 1—figure supplement 1B,C. A time-lapse video of a S2 cell coexpressing EB1-GFP and mCherry-CAMSAP3. Scale bar, 10 µm.
Video 4
Initial process outgrowth in Drosophila S2 cells is driven by microtubule minus-ends.
Related to Figure 1C. A time-lapse video of Drosophila S2 cells expressing GFP-CAMSAP3 plated for 5 min. Deep red dye was used to stain the membrane. Scale bars 10 µm and 5 µm, respectively.
Video 5
Microtubule minus-ends push the plasma membrane in growing neurites of young cultured Drosophila neurons.
Related to Figure 1I. Time-lapse video of a Drosophila neuron expressing elav>GFP-CAMSAP3 cultured for 4 hr. Deep red dye was used to stain the membrane. Scale bar 5 µm.
Video 6
Axonal microtubule organization switches from mixed to uniform orientation during neuron development.
Related to Figure 2A. Time-lapse videos of Drosophila-cultured neurons expressing ubi-EB1-GFP cultured for 4 hr, 21 hr and 48 hr. Scale bars, 10 µm.
Video 7
Dynein knockdown causes axons to contain antiparallel microtubules.
Related to Figure 2C. Time-lapse videos of a control and two elav>DHC shRNA Drosophila cultured neurons expressing EB1-GFP. Scale bars, 10 µm.
Video 8
Dynein RNAi causes mixed microtubule orientation in processes of Drosophila S2 cells.
Related to Figure 2F. Time-lapse videos of a control (untreated) and DHC RNAi Drosophila S2 cells expressing EB1-GFP. Scale bars, 10 µm.
Video 9
Cortical dynein sorts cytoplasmic microtubules.
Related to Figure 3F. Time-lapse images of S2 cells expressing GFP-CAMSAP3 cultured with 10 µM LatB. The drug was then washed out in control and DHC RNAi cells. Scale bars, 10 µm.
Video 10
Direct recruitment of cytoplasmic dynein to the plasma membrane activates its microtubule sorting activity in the absence of F-actin.
Related to Figure 3H. S2 cells expressing GAP43-FKBP, FBP-GFP-BicD and mCherry-CAMSAP3 (control and DHC RNAi) were cultured in the presence of 10 µM LatB for 2 hr. To recruit BicD to the membrane 1 …
