• Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration

      Blystone, Scott; Miller, Eric (2018)
      Metastasis is one of the leading causes of death in the world, affecting thousands every year. This is especially true of breast cancer, which can often result in the formation of secondary metastatic sites in the lung, liver, and bone marrow. There are many aspects to metastasis and an innumerable amount of molecular, biochemical, and cellular interactions contribute to its pathology. The ability of primary tumor cells to disseminate from the primary tumor, degrade the basement membrane, invade through the ECM, and eventually intravasate across the endothelial cell lining of the circulatory system or lymphatics requires a plethora of proteins, all working together in concert to achieve this. Nowhere in the cell is this more apparent than the actin cytoskeleton.Locomotion of cells requires several alterations in the actin cytoskeleton component of the cellular machinery. Generally speaking, cells must be able to polarize, form protrusions, adhere to the substratum, translocate, and then retract their tail, repeating this process as they continue to navigate to their destination. While there are many underlying aspects to this activity, spatiotemporal rearrangements of the actin cytoskeleton are key to the successful cellular motility. The mechanics behind dynamic actin cytoskeletal modifications are varied and complex, demonstrating the requirement for a variety of actin-associated, regulatory proteins.A crucial family of proteins involved in this process is the formin family of proteins. Formins are a relatively “new” group of actin modifiers which possess the unique ability to modify and generate linear actin filaments. While the members of this protein family all share some of the same actin modifying processes, many of these proteins also have functions exclusive to themselves. As a result, research into this field has blossomed and several novel features of different formins have been identified. Furthermore, alternative splice isoforms of several formins are often expressed in a variety of cell types, with specific functions attributed to each.The formin FMNL1 was originally identified in cells of a myeloid lineage and for many years was mostly thought to be involved in leukocyte adhesion and migration. Indeed, our lab has characterized many of the functions of this protein in both human and murine macrophages. However, as a result of the work in this dissertation, we have generated sufficient evidence suggesting that FMNL1 not only plays a role in breast cancer migration, but also exhibits functions unique to a specific alternative splice isoform of this protein.Our work on FMNL1 has pushed the field of study into this protein family in new directions. Herein, we have demonstrated that all three alternative splice isoforms of FMNL1 are expressed in a variety of cell types and the FMNL1ɣalternative splice isoform distinguishes itself from these isoforms via its ability to bundle linear actin filaments. Additionally, our data indicates that this is accomplished independently of the trademark FH2 domain, often thought to be the essential component of all formins. More specifically, we have identified a unique amino acid sequence in the C-terminal region of this isoform that most likely regulates this function. As a result, we have not only identified a potential therapeutic target for the treatment of metastasis via inhibition of cellular locomotion, but also pushed the field of formin research into a novel direction by providing insight which may foster new hypotheses and challenge classical theories regarding the relationship between formins and actin.
    • The Formin FMNL1 Contributes to the Macrophage Inflammatory Response by Regulating Podosome-dependent Adhesion and Migration.

      Blystone, Scott; Miller, Matthew (2015)
      Macrophages are indispensible white blood cells (leukocytes) that contribute to both the innate and adaptive immune response. They are crucial for resolving inflammatory events by clearing pathogens and cellular debris, in addition to promoting wound repair. Macrophages are derived from peripherally circulating monocytes, which after stimulation undergo diapedesis from the vasculature into the underlying complex extracellular matrix, where they can become fully differentiated macrophages and migrate to inflammatory loci. Tissues also contain residential populations of macrophages that aid in immediate immune responses and maintain tissue homeostasis. Conversely, unwarranted macrophage activation largely contributes to the onset and progression of inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, in addition to aiding cancer metastasis and facilitating organ transplant rejection. In order for macrophages to effectively resolve inflammatory events or contribute to disease pathology, they must be able to undergo directional migration, which is mediated by integrin-dependent adhesion complexes termed podosomes. Macrophage podosomes are the most prominent structure of the macrophage actin cytoskeleton, containing a pillar-like core of dense filamentous actin that is tethered to the cortical actincytoskeleton via radial actin filaments. Podosomes also contain a variety of proteins that are circumferentially arranged orassociated with the core, and thatare involved in signaling, linking, and scaffolding,as well as modulating the actin cytoskeleton.Historically, our lab has been interested in leukocyte integrin biology and understanding how these receptors mediate adhesion and migration through complex extracellular matrices. Previous studies in our lab demonstrated the novel podosomal association of an actin modulating protein with the ability to processively elongate unbranched linear actin filaments. Subsequent studies determined this protein to be the formin FMNL1, which is predominantly expressed in hematopoietic cells. Consequently, we further revealed that FMNL1 localizes to the apex of the dense actin core, and is required for podosome stability and macrophage adhesion.The work described in this dissertation has greatly expanded on these findings, demonstrating for the first time that primary macrophage migration is dependent on the formin FMNL1. Utilizing in vitro and in vivotechniques with aid of a novel conditional murine FMNL1 KO, we have observed that macrophage podosome formation, migration, and tissue distribution are dependent on FMNL1. Additionally, we have indicated that FMNL1 is required for embryonic development. Remarkably, our findings also suggest that FMNL1-dependent macrophage migration and podosome localization rely on the specific isoform FMNL1γ. Foremost, we have demonstrated that barbed end binding by the FMNL1γ FH2 domain is dispensable for its cellularfunction in macrophages, which has not been previously shown for any other cellular formin function. Thus, these findings, in addition to current formin knowledge, have allowed us develop a working model for FMNL1 function at macrophage podosomes. This work has distinguished FMNL1 as a unique therapeutic target to restrict macrophage migration that contributes to macrophage-mediated diseases. Furthermore, this could translate to treatment of certain cancers, since FMNL1 has been suggested to promote leukemic cell migration.