Sequestration of pRb by cyclin D3 causes intranuclear reorganization of lamin A/C during muscle cell differentiation

Abstract

The A-type lamins that localize in nuclear domains termed lamin speckles are reorganized and antigenically masked specifically during myoblast differentiation. This rearrangement was observed to be linked to the myogenic program as lamin speckles, stained with monoclonal antibody (mAb) LA-2H10, were reorganized in MyoD-transfected fibroblasts induced to transdifferentiate to muscle cells. In C2C12 myoblasts, speckles were reorganized early during differentiation in cyclin D3-expressing cells. Ectopic cyclin D3 induced lamin reorganization in C2C12 myoblasts but not in other cell types. Experiments with adenovirus E1A protein that can bind to and segregate the retinoblastoma protein (pRb) indicated that pRb was essential for the cyclin D3-mediated reorganization of lamin speckles. Cyclin D3-expressing myoblasts displayed site-specific reduction of pRb phosphorylation. Furthermore, disruption of lamin structures by overexpression of lamins inhibited expression of the muscle regulatory factor myogenin. Our results suggest that the reorganization of internal lamins in muscle cells is mediated by key regulators of the muscle differentiation program.

Figure 1.

Lamin speckles are reorganized in transdifferentiated fibroblasts. (A) NIH3T3 cells transfected with MyoD were transferred to DM for 48 h and then stained with LA-2H10 and antibodies to the muscle markers myogenin or myosin. One sample was extracted with detergent and nucleases before staining (Myosinext), and another sample was maintained in GM before staining with antibody to MyoD (MyoD-GM). (B) MyoD-transfected cells (in DM) were stained with a polyclonal antibody to lamins A/C (LA-P) and antibody to myogenin. (C) MyoD-transfected cells (in DM) were stained with LA-2H10 and antibodies to p21 and myogenin. Arrows indicate transdifferentiated cells not stained by LA-2H10 and an arrowhead points to an extracted cell showing recovery of LA-2H10 staining. Bar, 10 μm.

Figure 2.

Time course of expression of muscle markers and disappearance of lamin speckles. After transfer of C2C12 myoblasts to DM, samples were stained with LA-2H10 and antibodies to endogenous (A) cyclin D3 (CycD3), (B) MEF2C, or (C) GRIP-1 > 0–36 h. Arrow indicates a cyclin D3–positive cell at 12 h, which does not display lamin A/C speckles. DAPI staining of nuclei is shown in insets. Bar, 10 μm.

Figure 3.

Expression of ectopic cyclin D3 induces reorganization of lamin speckles. (A) C2C12 myoblasts were transfected with an HA-tagged cyclin D3 expression vector (in GM) and stained with HA antibody and mAb LA-2H10 or other indicated antibodies. Arrow indicates a transfected cell that does not display lamin A/C speckles. (B) A sample of transfected C2C12 cells was extracted with detergent and nucleases before staining with mAb LA-2H10 and HA antibody. Extracted cells were also stained with mAb LA-2B3. Arrowheads show transfected, extracted cells stained with LA-2H10. (C) C2C12 cells transfected with the cyclin D3 K112E mutant (CycD3m) were stained with LA-2H10 and HA antibody. (D) HeLa, C3H10T½, and NIH3T3 cells were transfected with the cyclin D3 construct and stained with LA-2H10 and HA antibody. DAPI staining of nuclei is shown in insets in A, C, and D. Bar, 10 μm.

Figure 4.

Ectopic GRIP-1 and MEF2C localize in speckles. C2C12 myoblasts were transfected with GRIP-1 and/or MEF2C expression vectors (in GM) and stained with antibodies to GRIP-1 or MEF2C, and LA-2H10. For cells transfected with both GRIP-1 and MEF2C, one sample was stained with GRIP-1 antibody and another with MEF2C antibody as indicated. Colocalized speckles are apparent as yellow spots in the merged panels. Similar data were obtained with antibodies to the epitope tags of GRIP-1 or MEF2C (unpublished data). Bar, 10 μm.

Figure 5.

Reorganization of lamin speckles requires pRb. (A) C2C12 myoblasts were transfected with cyclin D3 and/or E1A constructs (in GM) and stained with antibody to E1A and LA-2H10. Arrow indicates a cell transfected with cyclin D3 and E1A.928 (unable to bind to pRb), which does not show LA-2H10 staining. Similar results were obtained by using an antibody to the HA tag of cyclin D3 instead of E1A antibody. (B) Cells transfected with cyclin D3 and E1A (wild-type) were extracted with detergent and nucleases and stained (before and after extraction) with antibodies to E1A and HA. Arrows indicate the absence of cyclin D3 in extracted, transfected cells. (C) Myoblasts in GM were transfected with cyclin D3 or E1A constructs and counterstained with myogenin antibody. Arrowheads indicate myogenin-negative transfected cells. Areas enriched in myogenin-positive untransfected cells are displayed so as to provide positive controls for staining; only ∼5% of total untransfected myoblasts in GM were positive for myogenin. Bar, 10 μm.

Figure 6.

Cyclin D3 induces hypophosphorylation of pRb. Whole cell lysates of C2C12 myoblasts (Mb), myotubes (Mt), and cyclin D3–transfected myoblasts (tMb) in GM were analyzed by Western blotting with antibodies to pRbser780 and 795, HA tag of cyclin D3 (HACycD3), p21, myosin, lamin B1 (LB), and mab LA-2H10 (LA, LC). The phosphorylated species of pRb, which are dephosphorylated in myotubes and transfected myoblasts, are indicated by arrowheads.

Figure 7.

Loss of lamin speckles inhibits myogenin expression. (A) C2C12 myoblasts in GM were transfected with GFP-lamin A (GFP-LA), His-lamin A (His-LA), or GFP-N25lamin A (GFP-N25LA) and stained with lamin antibodies LA-2H10 or LA-2B3. His-lamin A–positive cells were stained with an antibody to the His epitope. Arrows indicate transfected cells that do not display lamin speckles. (B) Transfected myoblasts were transferred to DM and stained for myogenin after 36 h. Arrowheads point to transfected cells that do not express myogenin. Bar, 10 μm.

Figure 8.

Summary of factors regulating lamin reorganization in muscle cells. In C2C12 myoblasts, cyclin D3, either ectopically expressed in GM or endogenously in DM, and the hypophosphorylated form of pRb (pRb^*) produced under these conditions together mediate lamin reorganization. Expression of MyoD in NIH 3T3 cells in GM followed by transfer to DM to allow transdifferentiation to muscle cells also induces lamin reorganization. Differentiation and lamin reorganization are inhibited by E1A (by sequestration of pRb) and by the overexpression of lamin constructs that disrupt lamin structures. Other early differentiation markers such as myogenin, MEF2C, and GRIP-1 do not play a direct role in lamin reorganization.