LMP4 regulates Tbx5 protein subcellular localization and activity

Abstract

The limb- and heart-specific Tbx5 transcription factor coexpresses with and directly binds to the novel PDZ-LIM domain protein, LMP4. LMP4 is distributed in the cytoplasm associated with the actin cytoskeleton. In the presence of LMP4, Tbx5 shuttles dynamically between the nucleus and cytoplasm and, in a complex with LMP4, localizes to actin filaments. Nuclear and cytoplasmic Tbx5 distribution in developing chicken wings suggests the functional significance of the LMP4-Tbx5 interaction. In primary epicardial cells, we demonstrate that Tbx5 protein subcellular relocalization can be stimulated by external signals that induce cell differentiation. To test whether the relocalization from nuclear to cytoplasmic sites interferes with downstream gene expression, we used limb-specific Fgf10 and heart-specific Anf promoter-luciferase reporters and demonstrate that LMP4 acts as a repressor of Tbx5 activity. These studies reveal a previously unknown mechanism for Tbx transcription factor regulation in vertebrate limb and heart development and provide a better understanding of the molecular basis of hand/heart birth defects associated with Tbx5 mutations.

Figure 1.

Tbx5 and LMP4 protein localization in chicken wings. Serial cryosections of HH stage 36 wings were stained for Tbx5 (A and B) or LMP4 (C and D). (A and B) Tbx5 colocalized with nuclear marker DAPI (B) but was also localized to filamentous structures in the cytoplasm. (C and D) LMP4 (C) was localized to filamentous structures in the cytoplasm with no colocalization with DAPI (D). Comparison of merged images (B and D) shows a very similar staining pattern in the cytoplasm for Tbx5 and LMP4 in the wing. Bars, 10 μm.

Figure 2.

Localization of Tbx5 and LMP4 during epicardial cell differentiation. HH stage 25 chicken primary epicardial cells were cultured and stained for endogenous Tbx5 (A–H) or LMP4 (I–P) using specific antibodies. (A–D) Undifferentiated epicardial cells stained for Tbx5 (A), Alexa 488 phalloidin pseudocolored red (B), and the nuclear marker DAPI (C). Merged image (D) shows Tbx5 colocalization with DAPI but not with actin-stained phalloidin. (E–H) Epicardial cells induced to differentiate into EPDCs. Tbx5 localization (E) was nuclear but also displayed a filamentous cytoplasmic distribution. Merged image (H) shows Tbx5 colocalization with the nucleus and actin filaments. (I–L) Epicardial cells stained for LMP4 (I), actin (J), and DAPI (K). Merged image (L) shows colocalization of LMP4 with actin. (M–P) Differentiated EPDCs displayed filamentous and cortical localization of LMP4 (M), consistent with the reorganization of actin into stress fibers (N). No significant localization of LMP4 was detected within the nucleus (P). LMP4 also displayed nonfilamentous cytoplasmic localization in undifferentiated and differentiated epicardial cells (I and M). (Q–T) Differentiated EPDCs stained with anti-Tbx5 (Q), anti–LMP4-rhodamine (R), and DAPI (S). Merged image (T) shows significant colocalization of Tbx5 and LMP4 in the cytoplasm along actin filaments. EPDC differentiation was confirmed by the detection of calponin (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200511109/DC1). Bars, 20 μm.

Figure 3.

In single transfections, chicken Tbx5 and LMP4 localize to separate cellular compartments. (A–E) COS-7 cells transfected with Tbx5-HA and its expression detected using anti-Tbx5 antibodies (A). Cells were counterstained for actin using Alexa Fluor 633 phalloidin (B) and the nucleus using DAPI (C). The merged image (D) shows Tbx5 exclusively localized to the nucleus. Western blot of fractionated protein lysates from COS-7 cells transfected with Tbx5-HA, revealing exclusive nuclear localization of the Tbx5 protein (E). (F–J) COS-7 cells transfected with LMP4-myc and its expression detected using LMP4 antibodies (F). Cells were counterstained for actin (G) and the nucleus (H) as in B and C, respectively. The merged image (I) shows colocalization of LMP4 to actin stress fibers with no obvious nuclear localization. Western blot of fractionated protein lysates from COS-7 cells transfected with LMP4-myc, indicating localization of LMP4 to the cytoplasm (J). Empty vector transfections were used as controls for cellular fractionation. c, cytoplasmic fraction; n, nuclear fraction. Bars, 20 μm.

Figure 4.

In cotransfected cells, chicken Tbx5 and LMP4 interact at cytoplasmic sites. (A–D) COS-7 cells cotransfected with Tbx5-HA and LMP4-myc. Cells were stained with anti-HA for Tbx5 (A), anti-myc for LMP4 (B), and the nuclear stain DAPI (C). The merged image (D) shows colocalization of Tbx5 and LMP4 outside the nucleus, predominantly along actin fibers. Coimmunoprecipitation of Tbx5-HA and LMP4-myc from COS-7 protein lysates (E). LMP4-myc was immunoprecipitated with myc antibodies, and the Western blot was processed with Tbx5-specific antibodies. Protein molecular mass markers in kD are indicated on the left of the Western blot. Bar, 20 μm.

Figure 5.

Filamentous actin is required for cytoplasmic Tbx5–LMP4 complex localization. (A–E) COS-7 cells cotransfected with Tbx5-HA and LMP4-myc. 24 h after transfection, cells were treated with 2 μM latrunculin A for 60 min to sequester actin monomers. Cells were processed with anti-HA for Tbx5 (A), anti-myc for LMP4 (B), the nuclear stain DAPI (C), and Alexa Fluor 633 phalloidin to detect actin (E). The merged image (D) shows that the Tbx5–LMP4 complex no longer displays a filamentous pattern. For comparison, actin distribution of the cell in A–D is shown (E). (F) Coimmunoprecipitation of Tbx5 and LMP4 after actin disruption. Tbx5-HA was coprecipitated along with LMP4-myc in lysates from latrunculin A and DMSO control-treated COS-7 cells. Bar, 20 μm.

Figure 6.

LMP4 represses Tbx5 transcriptional activity on the Fgf10 and ANF target promoters. COS-7 cells were transfected with constant amounts of each respective reporter, Tbx5, and increasing amounts of LMP4 expression plasmid. Changes in luciferase reporter expression are indicated as fold activation. Asterisk indicates EGFP-Tbx5 fusion protein, revealing compromised activation of the Fgf10-luciferase reporter construct. Double asterisks indicate LMP4-EYFP fusion protein, revealing compromised repression of Tbx5 activity. Data shown are from two independent experiments performed in triplicate. Data are normalized to Renilla luciferase to control for differences in transfection efficiency.

Figure 7.

Dynamic shuttling of Tbx5 between nuclear and cytoplasmic compartments. COS-7 cells cotransfected with EGFP-Tbx5 and LMP4. (A) Photobleaching of cytoplasmic EGFP-Tbx5. Graph displays quantitative fluorescent data for a representative cell. (B) Photobleaching of nuclear EGFP-Tbx5. Graph displays representative quantitative fluorescent data. Bleaching of whole cell displayed no fluorescence recovery within the displayed time frame (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200511109/DC1). All FRAP assays were performed with a minimum of three cells.