Quantitative biological studies at cellular and sub-cellular level.

摘要

A magnetic tweezers is a device capable of exerting force of controlled amplitude on biological structures through transducers, typically paramagnetic beads. The first part of this dissertation describes in detail the development of a translational magnetic tweezers setup capable of generating both constant force pulses and complex force patterns of controlled amplitude in the range of 1-1000 pN over distances of hundreds of microns. The second part describes the biological applications that powered the magnetic tweezers development: quantitative biomechanical studies at cellular (extracellular) and sub-cellular (intracellular) level.;The extracellular studies addressed the process of membrane tether formation in eukaryotic cells. Tethers are nanotubular structures, which can be extracted out of the cell membrane by applying localized forces. We used the magnetic tweezers to extract simultaneously multiple tethers under constant force transduced to cells through magnetic beads attached to their membranes. The tethers were visualized by optical and scanning electron microscopy and quantitatively characterized in terms of membrane viscosity, elasticity and relaxation time. The contribution of the actin cytoskeletal network in the process of tether formation was also investigated. An interesting extension of this project focused on the verification of the Crooks fluctuation theorem (a fundamental recent finding in non-equilibrium thermodynamics) at the mesoscopic (i.e. membrane tether) level.;The intracellular studies involved the measurement of cytoplasmic viscoelastic coefficients of mouse oocytes using magnetic beads trapped into their cytoskeletal mesh. The motivation for this work was to improve on the success of oocyte cryopreservation, in particular on cryosurvival rate, by preserving the developmental competence of the eggs. We started out by quantifying the changes in the biophysical properties of the oocyte cytoskeleton induced by cryopreservation and found that cryopreservation altered all the viscoelastic parameters measurable with the tweezers. This finding inspired the hypothesis that the reversible disassembly of the actin cytoskeleton prior to cryopreservation prevents it from being damaged uncontrollably during freezing. We evaluated the effect of latrunculin A pretreatment on cryopreserved oocytes by using the viscoelastic parameters as markers of cytoskeletal integrity. The results showed that the relative number of oocytes that survived cryopreservation was higher in the latrunculin pretreated group. Moreover, the viscoelastic parameters in latrunculin treated survivors were similar to those of fresh oocytes. This finding promoted latrunculin A as a candidate drug for improving both cryosurvival and developmental competence in oocytes. The reversible dismantling of the actin cytoskeleton represents, conceptually, a novel approach to cryopreservation. The concept was also tested on somatic cells.