In Vivo Determination of Fluctuating Forces during Endosome Trafficking Using a Combination of Active and Passive Microrheology

Abstract

BackgroundRegulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology.Methodology/Principal FindingsThis dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking.Conclusions/SignificanceIn this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures.Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes.

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

“…This is not necessarily a drawback, as these symmetric bimodal distributions have never been clearly evidenced experimentally. Still, the stochastic model reproduces well that the tug-of-war leads to anomalous diffusion in agreement with some experimental observations [213,68,308,318]. Indeed, at very short time scales and if thermal fluctuations are taken into account, subdiffusion is observed as a result of the trapping of the cargo by oppositely pulling motors [193].…”

Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci

“…Furthermore, if thermal fluctuations are not the only driving force for intracellular motion, then the application of equilibrium theories, such as the Stokes-Einstein equation, to biological processes may not be valid. Indeed, several studies have demonstrated that the fluctuation-dissipation theorem is violated in vivo (4,5,7,8,42). These exciting findings may fundamentally change how we think about cellular processes and the nature of the cytoplasm (36).…”

Cytoskeleton and plasma-membrane damage resulting from exposure to sustained deformations: A review of the mechanobiology of chronic wounds

“…External cell measurements have been used to apply deformations and to measure local CSK and PM responses as well as whole cell mechanics. For example, methods such as atomic force microscopy [22,23] , magnetic [24,25] and laser tweezers [26,27] , have been used to apply forces at specific sites on the PM, inducing direct changes to the underlying actin CSK and revealing local PM dynamics and response to deformations. Whole cell stretching has been applied to adherent cells in two general approaches: those requiring cells to be suspended in solution [28] , and those where cells are adhered on a typically deformable substrate [9,10,[29][30][31][32][33] .…”