Crucial to a cell’s ability to migrate is the organization of its plasma membrane and associated proteins in a polarized manner to interact with and respond to its surrounding environment. Cells interact with the extracellular matrix (ECM) through specialized contact sites, including podosomes and invadopodia. Tumor cells use F-actin-rich invadopodia to degrade ECM and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction and degradation. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II,reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV increases the number of F-actin punctae, which are highly dynamic and co-localize with markers of podosomes and invadopodia. Endogenous SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases the average amount of matrix degradation; RNAi-mediated downregulation of SV decreases degradation. Cortactin, an essential component of both podosomes and invadopodia, binds SV sequences in vitro and contributes to the formation of EGFP-SV induced punctae. Additionally, SV affects cortactin localization,which could provide a mechanism for SV action at invadopodia. The formation of cholesterol-rich membrane rafts is one method of plasma membrane organization. A property of membrane rafts is resistance to extraction with cold Triton X-100 and subsequent flotation to low buoyant densities. The actin cytoskeleton has been implicated in many signaling events localized to membrane rafts, but interactions between actin and raft components are not well characterized. Our laboratory isolated a heavy detergent resistant membrane fraction from neutrophils, called DRM-H, that contains at least 23 plasma membrane proteins. DRM-H is rich in cytoskeletal proteins, including fodrin, actin, myosin II, as well as supervillin. DRM-H also contains proteins implicated in both raft organization and membrane-mediated signaling. DRM-H complexes exhibit a higher buoyant density than do most DRMs (referred to as DRM-L), which are deficient in cytoskeletal proteins. By using similar purification methods, I find that COS-7 cells also contain cytoskeleton-associated DRMs. In addition, when transfected into COS-7 cells, estrogen receptor (ER)α associates with DRM-H, while ERβ is seen in both DRM-L and DRM-H populations, suggesting a role for DRM-H in nongenomic estrogen signaling. Thus, the cytoskeleton-associated DRM-H not limited to hematopoietic cells and could constitute a scaffold for membrane raftcytoskeleton signaling events in many cells. Taken together, our results show that SV is a component of cytoskeleton-associated membrane rafts as well as podosomes and invadopodia, and that SV plays a role in invadopodial function. SV, with its connections to both membrane rafts and the cytoskeleton, is well situated to mediate cortactin localization, activation state, and/or dynamics of matrix metalloproteases at the ventral cell surface for proper matrix degradation through invadopodia. The molecular dissection of invadopodia formation and function may contribute to a greater understanding of in vivo invasion, and thus, tumor cell metastasis.

The family of runt related transcription factors (RUNX/Cbfa/AML/PEBP2) are essential for cellular differentiation and fetal development. RUNX factors are distributed throughout the nucleus in punctate foci that are associated with the nuclear matrix/scaffold and generally correspond with sites of active transcription. Truncations of RUNX proteins that eliminate the C-terminus including a 31-amino acid segment designated the nuclear matrix targeting signal (NMTS) lose nuclear matrix association and result in lethal hematopoietic (RUNX1) and skeletal (RUNX2) phenotypes in mice. These findings suggest that the targeting of RUNX factors to subnuclear foci may mediate the formation of multimeric regulatory complexes and contribute to transcriptional control. In this study, we hypothesized that RUNX transcription factors may dynamically move through the nucleus and associate with subnuclear domains in a C-terminal dependent mechanism to regulate transcription. Therefore, we investigated the subnuclear distribution and mobility of RUNX transcription factors in living cells using enhanced green fluorescent protein (EGFP) fused to RUNX proteins. The RUNX C-terminus was demonstrated to be necessary for the dynamic association of RUNX with stable subnuclear domains. Time-lapse fluorescence microscopy showed that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space in living cells. By measuring fluorescence recovery after photobleaching, both RUNX1 and RUNX2 were found to dynamically and rapidly associate with these subnuclear foci with a half-time of recovery in the ten-second time scale. A large immobile fraction of RUNX1 and RUNX2 proteins was observed in the photobleaching experiments, which suggests that this fraction of RUNX1 and RUNX2 proteins are immobilized through the C-terminal domain by interacting with the nuclear architecture. Truncation of the C-terminus of RUNX2, which removes the NMTS as well as several co-regulatory protein interaction domains, increases the mobility of RUNX2 by at least an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. Contributions of the NMTS sequence to the subnuclear distribution and mobility of RUNX2 were further assessed by creating point mutations in the NMTS of RUNX2 fused to EGFP. The results show that these point mutations decrease, but do not abolish, association with the nuclear matrix compared to wild-type EGFP-RUNX2. Three patterns of subnuclear distribution were similarly observed in living cells for both NMTS mutants and wild-type RUNX2. Furthermore, the NMTS mutations showed no measurable effect on the mobility of RUNX2. However, the mobility of RUNX proteins in each of the different subnuclear distributions observed in living cells were significantly different from each other. The punctate distribution appears to correlate with higher fluorescence intensity, suggesting that the protein concentration in the cell may have an effect on the formation or size of the foci. These findings suggest that the entire NMTS and/or the co-regulatory protein interaction domains may be necessary to immobilize RUNX2 proteins. Because RUNX factors contain a conserved intranuclear targeting signal, we examined whether RUNX1 and RUNX2 are targeted to common subnuclear domains. The results show that RUNX1 and RUNX2 colocalized in common subnuclear foci. Furthermore, RUNX subnuclear foci contain the co-regulatory protein CBFβ, which heterodimerizes with RUNX factors, and nascent transcripts as shown by BrUTP incorporation. These results suggest that RUNX subnuclear foci may represent sites of transcription containing multi-subunit transcription factor complexes. RUNX2 transcription factors induce expression of the osteocalcin promoter during osteoblast differentiation and to study both RUNX2 and osteocalcin function, it would be helpful to have transgenic mice in which OC expression could be easily evaluated. Therefore, to assess the in vivo regulation of osteocalcin by RUNX protein, we generated transgenic mice expressing EGFP controlled by the osteocalcin promoter. Our results show that EGFP is expressed from the OC promoter in a cultured osteosarcoma cell line, but not in a kidney cell line, and is induced by vitamin D3. Furthermore, the OC-EGFP transgenic mice specifically express EGFP in osteoblasts and osteocytes in bone tissues. Moreover, EGFP is expressed in mineralized bone nodules of differentiated bone marrow derived from transgenic mice. Thus, these mice produce a good model for studying the in vivo effects of RUNX-mediated osteocalcin regulation and for developing potential drug therapies for bone diseases. Taken together, our results in living cells support the conclusion that RUNX transcription factors dynamically associate with stationary subnuclear foci in a C-terminal dependent mechanism to regulate gene expression. Moreover, RUNX subnuclear foci represent transcription sites containing nascent transcripts and co-regulatory interacting proteins. These conclusions provide a mechanism for how RUNX transcription factors may associate with subnuclear foci to regulate gene expression. Furthermore, the OC-EGFP transgenic mice now provide a useful tool for studying the in vivo function and regulation of osteocalcin by RUNX proteins during osteoblast differentiation and possibly for developing therapeutic drugs for treatment of bone diseases in the future.