Lamin A and microtubules collaborate to maintain nuclear morphology

Abstract

Lamin A (LA) is a critical structural component of the nuclear lamina. Mutations within the LA gene (LMNA) lead to several human disorders, most striking of which is Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder. HGPS cells are best characterized by an abnormal nuclear morphology known as nuclear blebbing, which arises due to the accumulation of progerin, a dominant mutant form of LA. The microtubule (MT) network is known to mediate changes in nuclear morphology in the context of specific events such as mitosis, cell polarization, nucleus positioning and cellular migration. What is less understood is the role of the microtubule network in determining nuclear morphology during interphase. In this study, we elucidate the role of the cytoskeleton in regulation and misregulation of nuclear morphology through perturbations of both the lamina and the microtubule network. We found that LA knockout cells exhibit a crescent shape morphology associated with the microtubule-organizing center. Furthermore, this crescent shape ameliorates upon treatment with MT drugs, Nocodazole or Taxol. Expression of progerin, in LA knockout cells also rescues the crescent shape, although the response to Nocodazole or Taxol treatment is altered in comparison to cells expressing LA. Together these results describe a collaborative effort between LA and the MT network to maintain nuclear morphology.

Keywords: HGPS; lamin A; microbutuble; nuclear shape; progerin.

Figure 1.

Loss of LA results in an MTOC associated crescent shape. (A) Representative confocal immunofluorescence images of LA −/− and LA +/+ cells. Green = lamin B1; Blue = DAPI. (B) Frequency of crescent-shaped nuclei in LA−/− and LA+/+ cells. N > 100 nuclei per population. *** p <0.001.  Counted by 3 independent observers. Error bars are the standard deviation. (C) Representative confocal images of MTOC colocalization with crescent invagination in LA−/− cells. Green = gamma tubulin; Blue = DAPI. (D) Frequency of LA−/− and LA+/+ crescent-shaped nuclei colocalized with gamma tubulin. N >100 nuclei per population. Counted by 3 independent observers. Error bars are standard deviation. Error bars are the standard deviation. (E) Distribution of LA−/− and LA +/+ nuclear morphology as quantified by mean negative curvature. Density denotes cell count. N > 150. (F) Distribution of LA−/− and LA +/+ nuclear morphology as quantified by nuclear area. The area measurements are normalized to LA−/−. (G) Clustering of LA−/− and LA+/+ nuclei with a dotted line denoting the boundary between the 2 populations. Both metrics are normalized to LA−/−.

Figure 2.

Disruption of the microtubule network improves abnormal LA−/− nuclear morphology. (A) Representative confocal images of LA−/− and LA+/+ cells treated with Mock, 4µM Nocodazole (NOC), or 2µM Taxol (TX). Green = Lamin B1; Blue = DAPI. (B-D) Nuclear morphology quantifications of LA−/− and LA+/+ cells treated with Mock, 4μM NOC, and 2μM TX. (B) Mean negative curvature, (C) Area, (D) Volume measurements. N > 300 nuclei per population. Data is represented as mean with 95% confidence intervals. All values are normalized to LA−/− mock treated. *** p < 0.001. (E-G) Mean negative curvature by area contour plots. (E) LA−/− treated nuclei. (F) LA+/+ treated nuclei (G) Both LA−/− and LA+/+ nuclei excluding mock treated. N > 200 nuclei per population. Both metrics are normalized to LA−/− Mock. Dotted line depicts boundary between LA−/− and LA+/+. (H) Representative confocal images of LA−/− and LA+/+ cells treated with Mock, 4µM Nocodazole (NOC), or 2µM Taxol (TX). Red = Gamma-tubulin; Blue = DAPI.

Figure 3.

Ectopic expression of LA-GFP or Progerin-GFP partially rescues abnormal LA −/− nuclear morphology. (A) Representative confocal immunofluorescence images of LA −/− nuclei transfected with GFP, LA-GFP, or progerin-GFP. Green = GFP; Blue = DAPI. (B-C) Nuclear morphology quantification of GFP-expressing LA−/− nuclei. (B) Mean negative curvature, and (C) Area. N > 300 nuclei per population. Data is normalized to GFP-expressing LA−/− samples. *** p < 0.001. (D-E) Distribution of nuclear morphology quantification in LA−/− cells expressing GFP, LA-GFP or progerin-GFP.

Figure 4.

Progerin-GFP alters the response to cytoskeletal drugs. (A-C) Representative confocal immunofluorescence images of LA −/− nuclei transfected with (A) GFP, (B) LA-GFP, and (C) Progerin-GFP treated with either mock, 4μM NOC, or 2μM TX. Green = GFP; Red = Lamin B1; Blue = DAPI. (D-F) Quantification of transfected LA−/− nuclei (D) Mean negative curvature, (E) Area with Nocodazole treatment, and (F) Area with Taxol treatment. N >150 nuclei for each quantification. Data plotted as mean with 95% confidence intervals. All metrics are normalized to LA−/− GFP. **p < 0.01; ***p <0.001.

Figure 5.

Local Microtubule-Nucleus interaction. We hypothesize that the microtubule-organizing center (MTOC) locally deforms the nucleus mediated through LINC complexes. Force produced by the MTOC is normally resisted by the nucleus, specifically LA. Removal of LA leads to the invagination which can be rescued by progerin or LA expression. Nuclear morphology can also be rescued by Nocodazole and Taxol treatments which reduce MTOC force production.