Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts

Abstract

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

β-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells

“…Slides were cover-slipped using a mounting medium containing 4′6-diamidino-2-phenylindole (DAPI) in order to counterstain nuclei (F6057, Sigma). The micrographs of fluorescently labeled proteins were acquired using a confocal laser scanning microscope (CLSM) (TCS SP5; Leica, Microsystem GmbH, Mannheim, Germany) equipped as in Vergara et al (2015). DAPI fluorescent signal (in blue) was revealed with a 415–500 nm band pass filter, AF488-labeled secondary antibody (in green) was detected with a 495–519 nm band pass filter, and TRITC-phalloidin (in red) was evidenced with a 565–660 nm band pass filter.…”

Atomic force microscopy combined with optical microscopy for cells investigation

“…Vergara et al () investigated the contribution of parkin on the mechanical properties of the cells. In detail, by using parkin‐null fibroblast deriving directly from a Parkinson's patient, the authors studied the role of parkin to regulate the actin filament organization and the cellular stiffness.…”

“…This phenomenon happened because the release of the vesicle or the movement would require a local breakdown of the cytoskeleton with a considerable time and energy consumption increase. Vergara et al (2014) investigated the contribution of parkin on the mechanical properties of the cells. In detail, by using parkin-null fibroblast deriving directly from a Parkinson's patient, the authors studied the role of parkin to regulate the actin filament organization and the cellular stiffness.…”

Effects of Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) inhibition on sperm incorporation and cortical tension in mouse eggs

“…Together, these data indicate that UCH‐L1 affects certain features of the egg cortex and plasma membrane. The ubiqutitin system in general, and UCH‐L1 specifically, has been implicated in the function of actin‐associated processes and structures, including cortical tension (e.g., Bassères et al, ; Vergara et al, ). The data here support a model that the egg cortex in some way impacts the egg's responsiveness to a fertilizing sperm and/or conversion of the membrane to an unreceptive state after the first sperm has penetrated (McAvey et al, ).…”