Fig 1.
Localization of LICs in Mitosis.
A) Confocal immunofluorescence images of Hela cells depicting the localization of LIC1 (red) and LIC2 (green), shown by arrows. B) Fluorescence images of Hela cells showing the presence of LIC2 (green, using the ThermoScientific antibody) at kinetochores (CREST, red) in prometaphase. C) Fluorescence imaging after treatment of cells with anti-LIC2 siRNA, showing loss of LIC2 signal from spindle poles and kinetochores (arrow in inset). DAPI was used to visualize chromosomes. Scalebar is 5 μm in all images that are not zoomed.
Fig 2.
LIC2 is required for metaphase to anaphase progression.
A) Metaphase index (% of total cells present in metaphase +/- SD) in Hela cells treated with respective siRNAs as indicated. (3 experiments, n = approximately 500 cells per experiment). B) & C) Western blots showing siRNA mediated LIC1 and LIC2 specific knockdown. LIC2a and LIC2b represent two different siRNA sequences against human LIC2 [23]. The band marked by the arrow represents human LIC2 that gets reduced upon siRNA treatment, the upper bands are non-specifically recognized by the LIC2 antibody (ThermoScientific). Actin = loading control. D) Rescue of the LIC2-depletion induced metaphase arrest by transgenic expression of rat LIC2 (3 experiments, n = at least 500 cells per experiment). E) Western blots showing specific depletion of LIC2 upon treatment with LIC2-specific siRNAs, using a different LIC2 antibody (Abcam). LIC2a and LIC2b represent two different siRNA sequences against human LIC2 [23]. The band marked by the arrow represents human LIC2 that appears at the same molecular weight as in C.
Fig 3.
LIC2 depletion leads to prolonged metaphase arrest.
A) Still images from a movie featuring four different representative siRNA treated Hela cells (control, LIC2 and LIC 1 + 2) showing prolonged arrest in metaphase, followed by delayed anaphase onset (LIC2 > 80 min) or cell death (LIC2 > 4 hrs). B) Average metaphase to anaphase timing, (3 experiments, n = at least 100 cells per experiment). NEB = Nuclear Envelope Breakdown. Statistical significance was calculated by the T-test between two groups. C) Fraction of mitotic cells arrested for more than 80 minutes in metaphase upon respective siRNA treatment. (3 experiments, n = at least 100 cells per experiment). D) Fractions of metaphase cells dying after prolomged metaphase arrest (> 4 hrs) from the cells in C. p values in all panels are * p <0.05, ** p<0.01, *** p<0.001. Scalebar is 75 μm in all images as indicated.
Fig 4.
LIC2 acts on the spindle assembly checkpoint (SAC) to ensure mitotic progression.
A) Metaphase index in Hela cells after respective siRNA treatments of Mad2 and BubR1 alone or co-depleted with LIC2, as indicated on the x-axis (3 experiments, n = at least 200 cells per experiment). *** p<0.001, ns = not significant (p > 0.05). B) Western blot showing depletion of Mad2 by siRNA treatment. Actin = loading control.
Fig 5.
LIC2 depletion leads to increased inter-kinetochore tension in metaphase.
A) Confocal immunofluorescence images of metaphase Hela cells stained for kinetochores (red, CREST) and chromatin (DAPI). White lines indicate representative distance measurements between sister kinetochores. The insets on the right are zoomed to equivalent levels in both upper and lower panels. B) Average inter-kinetochore distance (μm) on y-axis. 3 experiments; n = at least 30 metaphase cells per experiment (10 pairs of sister chromatids measured per cell). ** = p < 0.01). Scalebar is 5 μm in all images that are not zoomed.
Fig 6.
LIC2 removes key SAC proteins from metaphase kinetochores to inactivate the spindle assembly checkpoint.
Representative images showing accumulation of SAC proteins A) Mad1, C) Zw10 and F) BubR1 at metaphase kinetochores upon prolonged metaphase arrest following indicated siRNA treatments. B, D, E, G) Left panels: integrated fluorescence intensities of SAC proteins normalized to the respective kinetochore (CREST) intensities for B) Mad1, D) Zw10, E) Mad2 and G) BubR1 at metaphase kinetochores. Y-axis = mean fluorescence intensity (+/- SEM) from 3 independent experiments, n = at least 18 metaphase cells per experiment for LIC1 and LIC2 depletion. Right panels: fold increase in normalized fluorescence intensity over control (GFP siRNA) for the various SAC proteins. Scalebar is 5 μm in all images.
Fig 7.
LIC2 biochemically interacts with key SAC proteins at metaphase.
A) Western blots of affinity purified mitotic LIC2 (LIC2-MTAP) probing for interaction of SAC proteins Mad1, Zw10 and BubR1 (right panel), and with the empty tag control (left panel). ß-actin was used as the loading control. Control indicates empty tag (MTAP vector alone) pulldown, LIC2 indicates LIC2-MTAP (see Materials and Methods). B) Upper panels: Immunoblots probing for the pulldown of dynein subunits DHC (dynein heavy chain) and IC-74 (dynein intermediate chain) upon affinity purification of the empty MTAP tag or LIC2-MTAP. Lower panels show successful pulldown of the respective bait proteins as positive controls for the affinity purification reaction. IN = input, FT = flow through, WS = wash, AP = affinity pulldown precipitate.
Fig 8.
A model for key LIC2 functions in SAC silencing during mitosis.
Novel functions of mitotic LIC2 uncovered from this study in silencing the spindle assembly checkpoint (SAC) are shown. LIC2-dynein strips attachment sensing SAC proteins (Mad1, Mad2, Zw10) from metaphase kinetochores, like LIC1-dynein. LIC2-dynein has the additional capability of causing removal of tension-sensing SAC protein BubR1, which is lacking in LIC1-dynein. The model offers a possible mechanistic explanation for the differential effects of the two LICs in mediating metaphase to anaphase progression.