835 S. Wolcott Ave., (M/C 901)
Chicago, IL 60612-7342
312-996-7620 phone
312-996-1414 fax

Primal de Lanerolle, PhD
Professor

Myosin I and II are members of a superfamily of actin-activated molecular motors that convert chemical energy into mechanical work. Most members of this superfamily do not form filaments.  Myosin I is the best studied of these "unconventional" myosins.  We discovered a nuclear isoform of myosin I that contains an unique 16 amino acid NH2-terminal extension.  Light and electron microscopy revealed that nuclear myosin I co-localizes with RNA polymerase I and II and biochemical and cell biology studies established that nuclear myosin I is essential for transcription by both polymerases.

Myosin molecules work in concert with actin and actin is also abundant in the nucleus. We have shown that actin is necessary for pre-initiation complex (PIC) formation, apparently because it facilitates the integration of the RNA polymerase II complex into the developing PIC. Interestingly, NMI is not needed for forming PICs, but it does appear to be necessary for forming the first few bonds during mRNA synthesis. Other experiments have shown that both actin and NMI stimulate transcription by RNA polymerase I and II.

We have also recently demonstrated that actin and nuclear myosin I are involved in chromosome movements in mammalian nuclei. The nucleus is organized into compartments and inactive or late replicating chromosomes are found near the nuclear envelope (heterochromatin in Fig. 1) while “active” chromosomes are found near the center of the nucleus. Previously, it was thought that the movement of chromosomes from one domain to the other occurred by a process called constrained diffusion. However, using wild type and mutant forms of actin and nuclear myosin I, we have shown that chromosome movement in an active process.

	Figure 1: Possible roles for actin and nuclear myosin I (NMI) in the nucleus. Actin binds tightly to RNA polymerase I and II while NMI binds to DNA. Thus, NMI heads sticking out from the DNA backbone could interact with actin bound to polymerases to function as a motor during transcription. NMI bound to DNA could also bind to short actin polymers to move active genes to the nuclear interior.

Our current research focuses on investigating the regulation of actin dynamics and analyzing the proteins that actin and nuclear myosin I bind to in the nucleus. Myosin V and myosin VI have also been discovered in the nucleus and we are studying how they, too, are involved in various nuclear functions. Myosin VI is especially intriguing because it moves in the opposite direction on actin filaments compared to all other myosins and it is likely to have unique functions in the nucleus.

Myosin II is also a major research focus in my lab. Actin and myosin II are major constituents of the cytoskeleton and, by generating force, they determine the physical characteristics of a cell.  The actin-myosin II interaction in smooth muscle and non-muscle cells is regulated by the phosphorylation of ser 19 of the 20 kDa light chain of myosin II by the enzyme myosin light chain kinase (MLCK). We, and others, have shown that myosin II phosphorylation is modulated by small G proteins (GTPases), such as Rho and Ras.  GTPases regulate a host of cellular responses and we have proposed that myosin II phosphorylation is an important part of the integrated cellular response to GTPase activation.

	Figure 2:  A high resolution analysis of the MLCK promoter using chloroacetaldehyde showed the formation of triplex H-DNA structures and that a mutation in SHR promoter forms a longer H-DNA structure than the promoter from normotensive rats.

We have used spontaneously hypertensive rats (SHR), a widely-used model of hypertension, to investigate this hypothesis. As with humans, blood pressure increases as SHR become older. In addition, hypertension in SHR is also highly dependent on the activation of the Ras signaling pathway by angiotensin II. We have shown that a mutation in the promoter increases MLCK expression and myosin light chain phosphorylation in SHR. We have also found that this mutation changes the structure of the MLCK promoter (Figure 2), making it more responsive to Ras signaling. Importantly, we have shown that inhibiting Ras signaling prevents the development of hypertension. We are now analyzing genomic DNA from hypertensive patients to determine if there are mutations that increase MLCK expression in human hypertension.