ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins
- King's College London School of Medicine, United Kingdom
- University of Utah School of Medicine, United States
Figures
Figure 1 with 1 supplement
ULK3 binds ESCRT-III via tandem MIT domains.
(A) Lysates from 293T cells overexpressing Myc-endosomal sorting complexes required for transport (ESCRT)-III proteins were mixed with lysates from cells non-transfected (−) or overexpressing …
-
Figure 1—source data 1
Data Collection and Refinement Statistics for the ULK3 MIT2:IST1 MIM1 Complex.
- https://doi.org/10.7554/eLife.06547.004
Figure 1—figure supplement 1
ULK3 binds to ESCRT-III via tandem MIT domains.
(A) Y2H assays in which ULK3 fused to the VP16 activation domain was tested for interactions with the designated human components of ESCRT-I, ESCRT-II, ESCRT-III, and ESCRT-associated proteins fused …
Figure 2 with 2 supplements
ULK3 regulates abscission timing.
(A) Asynchronous cultures of HeLa mCherry-Tubulin cells were transfected with the specified siRNA. Midbody resolution times were calculated from three separate experiments (mean times ±SD were …
Figure 2—figure supplement 1
ULK3 is not required for endosomal sorting or HIV-1 budding, but does regulate abscission timing.
(A) HeLa Kaposi sarcoma-associated herpesvirus (KSHV) K3 expressing cells were transfected with NT or two different siRNAs against ULK3 (ULK3-1 and ULK3-4). Cells were fixed and stained with a …
Figure 2—figure supplement 2
LAP2β-positive intercellular chromatin bridges resolve faster in ULK3-depleted cells.
(A) Selected frames from Videos 4, 5 showing the resolution dynamics of intercellular chromatin bridges present in HeLa cells expressing YFP-LAP2β transfected with NT siRNA (top) or ULK3 siRNA …
Figure 3
ESCRT-III binding and kinase activity are required for ULK3 function.
(A) HeLa mCherry-Tubulin cells stably expressing empty vector or siRNA-resistant ULK3 (ULK3R) were transfected with the indicated siRNA. Midbody resolution times were calculated in three separate …
Figure 4 with 1 supplement
Effects of ULK3 on IST1 localization at the midbody.
(A) HeLa mCherry-Tubulin cells stably expressing empty vector or ULK3 WT were stained with Hoechst and α-IST1 antibody. Cells connected by midbodies were classified by the localization pattern of …
Figure 4—figure supplement 1
Effects of ULK3 on the abscission machinery.
(A) HeLa cells stably expressing GFP-CHMP4B and empty vector, ULK3 WT, K44H, or M434D were fixed and stained with Hoechst, α-GFP, α-ULK3, and α-Tubulin antibodies. Cells connected by midbodies were …
Figure 5 with 1 supplement
Identification of ULK3 phosphorylation sites within IST1.
(A) In vitro kinase assay with recombinant ULK3 protein (middle) on immunoprecipitated HA-ESCRT-III proteins expressed in 293T cells. Western blot with α-HA antibody shows immunoprecipitated …
Figure 5—figure supplement 1
ULK3 phosphorylation of ESCRT-III proteins.
(A) 293T cells were co-transfected with vectors expressing Myc-ESCRT-III proteins and either empty vector, OSF-ULK3 WT, or OSF-ULK3 K44H. Cell lysates were electrophoresed on 10% Phos-tag gels and …
Figure 6 with 1 supplement
ULK3 phosphorylation of IST1 is required to sustain the abscission checkpoint and inhibits IST1 function in abscission.
(A) HeLa cells stably expressing empty vector, IST1R WT, or IST1R 4SA were co-transfected with the indicated siRNA and NUP153 siRNA to trigger the abscission checkpoint. Cells were fixed and stained …
Figure 6—figure supplement 1
The IST1 phosphorylation 4SA mutant retains IST1 binding and abscission functions.
(A) Y2H assays with IST1 WT and IST1 4SA fused to the VP16 activation domain showing binding to VPS4A, VPSP4B, CHMP1A, CHMP1B, LIP5, and MITD1 proteins fused to the Gal4 DNA-binding domain. CHMP2A …
Figure 7 with 1 supplement
ULK3 and CHMP4C are functionally interconnected within the abscission control pathway.
(A) HeLa cells expressing GFP-CHMP4C were transfected with NT, ULK3, or CHMP4C siRNA, fixed and stained with Hoechst and α-Tubulin antibody to visualize multinucleated and cells connected by …
Figure 7—figure supplement 1
ULK3 phosphorylates CHMP4C.
(A and B) Lysates from 293T cells expressing HA-CHMP4 constructs were immunoprecipitated with α-HA antibodies and subjected to in vitro kinase assays with recombinant ULK3. Incorporated ATP γ32P was …
Author response image 1
Author response image 2
Videos
Video 1
Representative example of asynchronous HeLa mCherry-Tubulin cells treated with non-targeting (NT) siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 90 min. Related to Figure 2 and Figure 2—figure supplement 1.
Video 2
Representative example of asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 60 min. Related to Figure 2 and Figure 2—figure supplement 1.
Video 3
Representative example of asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 60 min. Related to Figure 2 and Figure 2—figure supplement 1.
Video 4
Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β transfected with NT siRNA.
Chromatin bridge resolution time is 510 min. Bridge resolution is indicated with an arrow. Related to Figure 2 and Figure 2—figure supplement 2.
Video 5
Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β transfected with ULK3 siRNA.
Chromatin bridge resolution time is 110 min. Bridge resolution is indicated with an arrow. Related to Figure 2 and Figure 2—figure supplement 2.
Video 6
Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β and mCherry-Tubulin, containing an intercellular chromatin bridge that resolves in 850 min when the daughter cells faithfully divide.
Bridge resolution is indicated with an arrow. Related to Figure 2—figure supplement 2.
Video 7
Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β and mCherry-Tubulin, containing an intercellular chromatin bridge that does not resolve and the cleavage furrow regresses.
Related to Figure 2—figure supplement 2.
Video 8
Representative example of low-density asynchronous HeLa mCherry-Tubulin cells treated with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 170 min. Related to Figure 2.
Video 9
Representative example of high-density asynchronous HeLa mCherry-Tubulin cells treated with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 60 min. Related to Figure 2.
Video 10
Representative example of low-density asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 60 min. Related to Figure 2.
Video 11
Representative example of high-density asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 60 min. Related to Figure 2.
Video 12
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 V338D.
Midbody resolution is indicated with an arrow. Abscission time is 80 min. Related to Figure 3.
Video 13
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 M434D.
Midbody resolution is indicated with an arrow. Abscission time is 70 min. Related to Figure 3.
Video 14
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 K44H.
Midbody resolution is indicated with an arrow. Abscission time is 80 min. Related to Figure 3.
Video 15
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and empty vector.
Midbody resolution is indicated with an arrow. Abscission time is 100 min. Related to Figure 3.
Video 16
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 WT.
Midbody resolution is indicated with an arrow. Abscission time is 160 min. Related to Figure 3.
Video 17
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 WT.
Midbody resolution is indicated with an arrow. Abscission time is 290 min. Related to Figure 3.
Video 18
Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and empty vector transfected with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 90 min. Related to Figure 6.
Video 19
Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and empty vector transfected with IST1 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 190 min. Related to Figure 6.
Video 20
Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and siRNA-resistant IST1 (IST1R) WT transfected with IST1 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 90 min. Related to Figure 6.
Video 21
Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and IST1R 4SA transfected with IST1 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 90 min. Related to Figure 6.
Video 22
Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and IST1R 4SE transfected with IST1 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 180 min. Related to Figure 6.
Video 23
Representative example of asynchronous HeLa cells stably expressing GFP-CHMP4B and mCherry-Tubulin transfected with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 110 min. Related to Figure 7.
Video 24
Representative example of asynchronous HeLa cells stably expressing GFP-CHMP4C and mCherry-Tubulin transfected with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 140 min. Related to Figure 7.
Video 25
Representative example of asynchronous HeLa cells stably expressing GFP-CHMP4C and mCherry-Tubulin transfected with ULK3 siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 80 min. Related to Figure 7.
Video 26
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and empty vector transfected with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 80 min. Related to Figure 7.
Video 27
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 transfected with NT siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 120 min. Related to Figure 7.
Video 28
Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 transfected with CHMP4C siRNA.
Midbody resolution is indicated with an arrow. Abscission time is 90 min. Related to Figure 7.
Additional files
-
Supplementary file 1
Supplementary information. Includes list of plasmids used in this study, siRNA sequences, primary antibodies, stable cell lines used in this study.
- https://doi.org/10.7554/eLife.06547.046
