טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZattelman Lilach
SubjectFrom Enzymatic Adaptation to Cellular Function, How Myosin
1C Isoforms Impact Nucleolus Structure
and Function
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Professor Arnon Henn


Abstract

The nucleolus has emerged as hub for regulating and integrating fundamental and key cellular processes during cellular homeostasis and consequently disfunction in pathophysiological conditions. In addition, and independently of its primary function in ribosome biogenesis, these extra-nucleolar activities termed as “non-canonical” are integrated into many cellular pathways and hence elevate the level of complexity of nucleolus biology. The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which bind PtdIns(4,5)P2 through a pleckstrin homology (PH) domain in their tail, exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear-cytoplasmic partitioning. MYO1C splice-isoforms, an actin-based molecular motors, were shown to play a role in RNA polymerase I transcription; however, the spatial and temporal aspects were neglected as well as its molecular mechanism of action and how its force production hallmark is being utilized. To gain temporal insights on MYO1C role in the nucleolar linked cellular homeostasis, we have conditionally knocked down (KD) all three MYO1C isoforms. As a result, both ribosome structure and function were affected. In addition to a concentration dependent reduction in rRNA transcription, when MYO1C protein levels were lower than two-folds, the cells show morphological changes and almost complete reduction in proliferation rate. Systematic and quantitative analysis of the nucleolar numbers and morphology revealed that 16 hours post MYO1C KD induction, 60% of the cells show nucleolar disassembly, followed by cell cycle G0/G1 arrest 54 hours post induction. Thus, we hypothesize that MYO1C function in the nucleolus affects both the canonical and non-canonical nucleolar functions.

The gap in knowledge of the isoforms’ ATPase activity to this date has paradoxically created an opportunity to utilize their enzymatic adaptation to reveal the hidden springs that may provide contractile activity in the aforementioned phenotype. Therefore, we first established the basis to test this hypothesis by investigating the isoforms’ activities by a comparative approach. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms. Biophysical studies reveal that the NTRs regulate the nucleotide-binding pocket isomerization of the MYO1C isoforms, and hence modulate their nucleotide binding properties. Structural studies showed steric effect of the NTR on the nucleotide binding pocket isomerization and the power stroke mechanism of MYO1C. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, which broadens their ability to work under different loads. We propose that MYO1C isoforms have evolved a tension sensing mechanism between PtdIns(4,5)P2 membrane structures and actin scaffold adapted for their unique cellular functions within plasma membrane, nucleus and nucleolus. Specifically, we propose that MYO1C16 has evolved to work in the super crowded environment of the nucleolus’s dense fibrillar center, as a contractile machinery with a spectrum of adjustable springs while bound to PtdIns(4,5)P2 and actin. The specific function of each isoform may prove highly unique based on our enzymatic studies that is yet to be explored.