The main focus of the lab is to understand the roles of myosins in the nucleus and their effects upon transcription. The presence of myosins in the nucleus is well established but what role they play in the organisation and transcription of genetic information is unclear. For the first time we combine classical biochemical and single molecule assays to provide a quantitative high resolution description of the activity of these nuclear myosins; providing a mechanistic insight into the activity of these proteins.
Through this approach it will not only be possible to identify what is the function of the motors, but also how they function. We focus upon two contrasting myosin motors: Nuclear Myosin I and Myosin VI.
Myosins are actin binding proteins which are implicated in a variety of cellular functions. The proteins consist of a conserved motor domain with ATP and actin binding ability. This is followed by a neck/lever arm which contains IQ motifs for calmodulin (CaM) binding, and finally a tail domain (Fig. 1). Myosin diversity is related to the tail domains which allow regulation, dimerization, cargo and lipid binding.
Fig. 1 (A) Nuclear Myosin I Tag shown in blue. Nuclear localisation sequence (NLS) is located in the CaM binding IQ motifs. A lipid binding PH domain is in the Tail. (B) Myosin VI Tail contains a short coiled-coil and lipid binding site (PH/PIP2).
This study involves investigating myosin away from the traditional interactions with filamentous actin (F-Actin), as seen with muscle contraction and cellular transport. The existence of myosin and actin in the nucleus is a relatively new discovery.
Nuclear ActinNuclear actin is predominantly monomeric (b-Actin) but can exhibit a polymeric behaviour (Schoenenberger et al 2005). This immediately poses interesting and fundamental questions regarding the nature of the interaction between nuclear myosin and actin. Do they interact? Can they generate force? How could this force be generated? Actin interacts with RNA Polymerase II (RNAPII) (main cell polymerase), increasing its activity at least 8-fold and is therefore considered a transcription factor (TF) (Hofmann et al 2004; Grummt 2006). TFs are vital regulators of RNAPs, dictating development, stem cell differentiation and cell responses. RNAPII consists of 12 subunits which bind to short DNA sequences containing the TATA-box situated 20-30 bases upstream of the transcription-start-site. TFs associate with RNAPII in this region to activate transcription (Myer and Young 1998).
Nuclear Myosin INuclear myosin I (NMI), an isoform of myosin Ic, was the first discovered myosin in the nucleus (Fig. 1A). NMI has a 16 amino acid N-terminal tag, which is required for nuclear localization but it is not the traditional nuclear localization sequence (Pestic-Dragovich et al 2000) which is located within the IQ domains. With regard to transcription, the ATPase activity is required for transcription initiation and forming the first phosphodiester bond (Philimonenko et al 2004). This means that myosins can also be considered a TF (Hofmann et al 2006b).
Fig. 2: Possible roles of NMI. NMI binds b-Actin on the RNAPII, while being bound to DNA. A second NMI associates with the complex to aid translocation during elongation.