Combined strategy of siRNA and osteoclast actin cytoskeleton automated imaging to identify novel regulators of bone resorption shows a non-mitotic function for anillin

Abstract

Osteoclasts are the main cells responsible for the resorption of mineralized extracellular matrices. They are the major targets for anti-resorptive therapies to manage osteoporosis, a major public health problem. Osteoclasts are giant multinucleated cells that can organize their a unique adhesion structure based on a belt of podosomes, which is the keystone of the bone resorption apparatus. We combined differential transcriptomics and siRNA screening approaches to get a broader view of cytoskeletal regulators that participate in the control of osteoclast cytoskeleton and identify novel regulators of bone resorption by osteoclasts. We identified 20 new candidate regulators of osteoclasts cytoskeleton including Fkbp15, Spire1, Tacc2 and RalA, for which we confirmed they are necessary for proper organization of the podosome belt. We also showed that Anillin, well known for its role during cytokinesis, is essential in osteoclasts for correct podosome patterning and efficient bone resorption. In particular, Anillin controls the levels of the GTPase RhoA, a known regulator of osteoclast cytoskeleton and resorption activity. Finally, we set up and validated an automated imaging strategy based on open-source software for automatic and objective measurement of actin cytoskeleton organization in osteoclasts. We provide these pipelines that are useful to automatically assess the effect of collections of siRNAs or chemical compounds on osteoclast cytoskeleton or differentiation. 4Introduction.

The osteoclast cytoskeleton – current understanding and therapeutic perspectives for osteoporosis

“…2). Differential gene expression has been used to identify the regulators of RhoGTPase activity necessary for osteoclast cytoskeleton organization and efficient bone resorption (Brazier et al, 2006;Georgess et al, 2014b;Maurin et al, 2018). The expression of various Rho guanine nucleotide exchange factors (GEFs), which activate the RhoGTPases by allowing their binding to GTP (Fort and Blangy, 2017), increases during osteoclast differentiation (Brazier et al, 2006).…”