JCS045377 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Figure S1. Overexpression of nonfunctional KIF3b fragments interferes with p0071 midbody localization. HeLa cells were transfected with nonfunctional KIF3b fragments, then synchronized and stained for p0071 as indicated. The expression of nonfunctional N-terminal and C-terminal KIF3b fragments was monitored by fluorescence of the N-terminal RFP tag. Both KIF3b fragments prevented p0071 accumulation at the midbody. Scale bars: 10 µm, enlargements 5 µm.

• Supplemental Figure S2 -

  Fig. S2. A KIF3b-binding-deficient mutant of p0071, p0071Δrep1, does not localize at the midbody. HeLa cells were transfected with wild-type p0071-DsRed (A) or p0071Δrep1-DsRed (B) and stained for α-tubulin and DNA. (A) Wild-type p0071 associated with intercellular junctions in interphase cells (upper panel) as observed in MCF-7 cells. During cytokinesis wild-type p0071 accumulated around the central microtubule bundle (lower panel). (B) As shown in MCF-7 cells, p0071Δrep1 exhibited plasma membrane association in interphase cells (upper panel) but was mislocalized during cytokinesis (lower panels). Scale bars: 20 µm, enlargements 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Localization of wild-type p0071, p0071Δrep1 and p0071−MKLP1-motor proteins. HEK293 cells were transfected with control siRNA (left panel) or KIF3b siRNA (right panel) plus the p0071 constructs as indicated and stained for α-tubulin and DAPI. Wild-type P0071 revealed decreased labeling at the midzone after KIF3b knockdown compared with control siRNA-transfected cells. The p0071Δrep1 mutant protein failed to accumulate at the midbody in control and KIF3b siRNA-transfected cells. The p0071−MKLP1-motor fusion protein accumulated at the microtubule midzone in anaphase as is typical for the MKLP1 motor. This localization persists after KIF3b knockdown. Scale bars: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Motor proteins involved in transport of Rho signaling components during cytokinesis. Motor proteins are depicted in green, their direct cargo proteins in blue. The par complex par3-par6-aPKC has not been validated in cytokinesis. RhoA (pink) and MLC (yellow) were not directly transported by the depicted motor proteins. Red lines indicate interactions between the cargo proteins.