TY - JOUR

T1 - Calmodulin downregulation in conditional knockout HeLa cells inhibits cell migration

AU - Alcalde, Juan

AU - Munk, Mads

AU - González-Muñoz, María

AU - Panina, Svetlana

AU - Berchtold, Martin W.

AU - Villalobo, Antonio

PY - 2021

Y1 - 2021

N2 - The study of calmodulin (CaM) functions in living cells has been tackled up to date using cell-permeant CaM inhibitors or interference-RNA methods. CaM inhibitors may lack specificity and the siRNA interference approach is challenging, as all three CaM genes expressing an identical protein in mammals have to be blocked. Therefore, we recently introduced a novel genetic system using CRISPR/Cas9-mediated gene deletion and conditional CaM expression to study the function of CaM in HeLa cells. Here, we describe the effect of CaM downregulation on the basal and epidermal growth factor (EGF)-dependent 2D- and 3D-migration in HeLa cells. CaM downregulation inhibited cell migration on a 2D-surface in the absence but not in the presence of EGF. In contrast, CaM downregulation led to inhibition of 3D-migration across a porous membrane both in the absence and presence of EGF. CaM downregulation decreased the expression of Rac1, Cdc42 and RhoA, all known to play crucial roles in cell migration. These results show that EGF-dependent 2D- and 3D-migration utilize distinct CaM-regulated systems and identify several essential migratory proteins directly or indirectly regulated by CaM.

AB - The study of calmodulin (CaM) functions in living cells has been tackled up to date using cell-permeant CaM inhibitors or interference-RNA methods. CaM inhibitors may lack specificity and the siRNA interference approach is challenging, as all three CaM genes expressing an identical protein in mammals have to be blocked. Therefore, we recently introduced a novel genetic system using CRISPR/Cas9-mediated gene deletion and conditional CaM expression to study the function of CaM in HeLa cells. Here, we describe the effect of CaM downregulation on the basal and epidermal growth factor (EGF)-dependent 2D- and 3D-migration in HeLa cells. CaM downregulation inhibited cell migration on a 2D-surface in the absence but not in the presence of EGF. In contrast, CaM downregulation led to inhibition of 3D-migration across a porous membrane both in the absence and presence of EGF. CaM downregulation decreased the expression of Rac1, Cdc42 and RhoA, all known to play crucial roles in cell migration. These results show that EGF-dependent 2D- and 3D-migration utilize distinct CaM-regulated systems and identify several essential migratory proteins directly or indirectly regulated by CaM.

KW - Calmodulin

KW - Cdc42

KW - Cell migration

KW - Conditional knock-out cells

KW - Rac1

KW - RhoA

U2 - 10.1016/j.abb.2020.108680

DO - 10.1016/j.abb.2020.108680

M3 - Journal article

C2 - 33220265

AN - SCOPUS:85097427131

VL - 697

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

M1 - 108680

ER -