Figure 2.

KIFC3 Localizes to Dendrites and Interacts with CAMSAP2

(A and B) Representative images of DIV11 hippocampal neurons transfected with mCherry-KIFC3-FL and co-stained with acetylated tubulin and tyrosinated tubulin antibody.

(A) Selected axonal (marked with a blue box) and dendritic (marked with an orange box) regions are enlarged below. Scale bars, 10 μm and 5 μm in enlargements.

(B) Further enlargement of a dendrite region is shown. Scale bars, 2 μm.

(C) Intensity profile of the indicated region in (B).

(D–F) Representative images of DIV10 hippocampal neurons transfected with GFP fill and mCherry-KIFC3-FL (D), mCherry-KIFC3-N1 (E), or mCherry-KIFC3-N2 (F) and stained for TRIM46 to visualize the axon initial segment (AIS). Selected axonal (marked with a blue box) and dendritic (marked with an orange box) regions are enlarged below. Scale bars, 10 μm.

(G) Schematic representation of the KIFC3 secondary structure and truncation constructs used. CC, coiled-coiled domain; MD, motor domain.

(H) Polarity index analysis from KIFC3-FL and truncations corresponding to (D)–(F). Positive values indicate axon enrichment, and negative values indicate dendrite enrichment. Error bars, SEM. N = 2. KIFC3-FL: n = 12, KIFC3-N1: n = 10, KIFC3-N2: n = 9, and KIFC3-N5: n = 6.

(I) Biotin pull-downs from extracts of HEK293T cells transfected with BirA recognition site conjugated mCherry-KIFC3-FL and probed for mCherry and CAMSAP2. For all pull‐down experiments, the input is 25% of the biotin pull-down. CAMSAP2 was found from KIFC3 pull-downs with brain extracts (Table S1). The interaction is also proved by co-localization experiment in neurons and COS7 cells (Figure S2; Video S1).

(J) Biotin pull-downs from extracts of HEK293T cells transfected with BirA recognition site conjugated mCherry-KIFC3-FL together with GFP-tagged CAMSAP1, 2, or 3 and probed for mCherry and GFP.

(K) Biotin pull-downs from extracts of HEK293T cells transfected with BirA recognition site conjugated KIFC3 truncation constructs together with GFP-tagged CAMSAP2 and probed for mCherry and GFP.

(L) Representative images of COS7, transfected with 3GFP-CAMSAP2 and mCherry-KIFC3 (Video S2). Silicon rhodamine (SIR)-tubulin was applied to visualize microtubules. Microtubule photoablation was indicated by red stars. Minus end was indicated by white and black arrows. 3GFP-CAMSAP2 was shown in green, mCherry-KIFC3 in red, and SIR-tubulin in cyan. Scale bars, 2 μm.

(M) The quantification of CAMSAP2 and KIFC3 puncta intensity corresponding to (L).