Investigating the mechanomolecular determinants governing the translocation of proteins into the cell nucleus across length scales

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Cells in all living organisms have the ability to sense the physical properties of the extracellular environment and adapt accordingly by changing their function. The translation of mechanical cues into biochemical signals is termed mechanotransduction. Several proteins, called transcription factors, that function as activators (or repressors) of gene expression, are mechanosensitive. In general, mechanosensitive transcription factors reside in the focal adhesions / cytoplasm, however upon mechanical stimulation can shuttle into the cell nucleus across the Nuclear Pore Complex (NPC), which is the main gateway in and out from the nucleus. We recently discovered that the nuclear translocation across the NPC is mechanoselective. However, we still do not understand the molecular mechanisms that underpin the mechanoselectivity of the NPC.

In this thesis, I first used single cell and single molecule nanomechanical approaches to initially investigate how proteins residing in different cell compartments (i.e. zyxin – found in focal adhesions and myocardin-related transcription factor A; MRTFA – shuttling between the cytoplasm and the nucleus) are regulating their translocation within different cellular compartments upon external mechanical stimuli. Secondly, following our recent evidence unveiling a correlation between a protein’s mechanical stability and its nuclear import dynamics, I set out to test whether the nanomechanical rules observed for proteins in vitro in single molecule nanomechanical experiments are directly translated to the behaviour of the same proteins when translocating to the nucleus through the NPC in the cellular environment. To answer this question, I first used single molecule force spectroscopy AFM and magnetic tweezers to characterise the mechanical properties of the zinc-finger-rich C-terminus of zyxin, which is known to shuttle from the focal adhesions to the nucleus. This revealed the reversibility of the zinc fingers after dissociation and their ability to form under applied force. At the cellular level, I explored how zyxin changes its localisation when the cell is exposed to different levels of mechanical stress, achieved by e.g. seeding cells on gels of varying rigidity. Zyxin’s nuclear presence found to be increased when cells were seeded on matrices of low stiffness, while a positive correlation between actin polymerisation and zyxin cytoplasmic localisation was drawn. Secondly, and within a conceptually similar approach, I studied the mechanoselectivity of the NPC by using optogenetic tools that regulate nuclear localisation tagged with proteins of different mechanical stabilities, independently assessed in vitro using single molecule nanomechanical techniques. In particular, I addressed (a) the molecular mechanisms that underlie the mechanoselectivity of the nuclear pore (involvement of Nup153) and (b) the interplay between mass and mechanical stability in regulating nuclear entry (mechanical stability of cargo dominated its nuclear passage upon a ~ 90 kDa mass threshold) to (c) engineer increased nuclear mechanotransduction of transcription factors through molecular design (by addition of short peptide-tag). I finally tested the functional implications of these molecular modifications at the cellular level. Combined, these experiments lay the foundation for our understanding of the molecular determinants that govern nuclear mechanotransduction across the NPC.

Date of Award1 Dec 2022
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorSergi Garcia-Manyes (Supervisor) & Katelyn Spillane (Supervisor)

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