Fast Fluorescence Laser Tracking Microrheometry II: Quantitative Studies of Cytoskeletal Mechanotransduction

¹ MIT
² Brigham & Women's Hospital

^* To whom correspondence should be addressed. E-mail: maxine.jonas{at}gmail.com.

Submitted on August 21, 2007
Revised on October 10, 2007
Accepted on 18 March 2008

	Abstract

Fluorescence laser tracking microrheometry (FLTM) is a novel method able to assess the local, frequency-dependent mechanical properties of living cells with nanometer spatial sensitivity at speeds up to 50 kHz. In a companion article [1], we described the design, development and optimization phases of the FLTM before reporting its performances in a variety of viscoelastic materials. In the present work, we demonstrated the suitability of FLTM to study local cellular rheology and obtained values for the storage and loss moduli G'() and G''() of fibroblasts consistent with past literature. We further established that chemically-induced cytoskeletal disruption is accompanied by reduced cellular stiffness and viscosity. Next, we provided a systematic study of some experimental variables that may critically influence microrheology measurements. First, we interrogated and justified the relevance of bead endocytosis as a method of cellular internalization of 1-µ probes in FLTM. Second, we showed that as sample temperature increased, FLTM findings were elevated towards higher frequencies. Third, we confirmed that relevant bead sizes (1 and 2 µm) had no effect on FLTM measurements. Fourth, we report the lack of influence of bead coatings (anti-integrin, anti-transferrin, anti-dystroglycan, or uncoated tracers were surveyed) on their rheological readouts. Finally, we demonstrate the potential of FLTM in studying how substratum rigidity regulates cellular rheological properties. Interestingly, multiple, coupled strain relaxation mechanisms can be observed separated by two plateau moduli. While these observations can be partly explained by rheological theories describing entangled actin filaments, there is a clear need to extend existing microrheology models to the cytoskeleton, including potentially important factors such as network geometry and remodeling.

Key Words: Brownian motion, cytoskeleton, high resolution, mechanotransduction, rheology