Targeting membrane repair processes in cancer cells

Hovedområde:Cell biology
 
Målgruppe:Biochemistry, Molecular Biomedicine, Biology
 
Niveau:Masters
 
Projektbeskrivelse:

Plasma membrane repair is a fundamental process essential for cell survival upon membrane damage (1). Metastazising cancer cells need to have especially efficient membrane repair systems to cope with continuous mechanical stress leading to membrane damage.  All membrane repair mechanisms are Ca2+ dependent. How the Ca2+ signal is used in plasma repair mechanisms is not well understood. Our aim is to investigate the role of Ca2+ and Ca2+ -handling/signaling processes in membrane damage and repair. The Ca2+-binding protein ALG-2 has been found to be implicated in the "macro vesicle shedding" mechanism by mediating the assembly of the ESCRT machinery at membrane wound sites. Recent data show that the Ca2+ binding protein ALG-2 is essential for membrane repair after damaging myocytes by laser treatment (2). Our recently published results with a chicken ALG-2 knock out cell line show that these cells recover less well than the parental wild type cells following electroporation - induced membrane damage and that this phenotype can be reversed by ectopically expressing ALG-2 (3). Now, we have created knock out HeLa cancer cells by CRISPR/Cas9 lacking ALG-2 and the related protein Peflin, which dimerizes with ALG-2. We will test different ALG-2 constructs, among others constructs with inactivated Ca2+ binding sites to check for the Ca2+ requirement of the ALG-2-mediated process for their membrane repair potential after exposing cells to plasma membrane damage. Membrane damage will be induced by digitonin, streptolysin O and other membrane damaging reagents/treatments followed by the uptake of dyes via flow cytometry. In addition, we will investigate the role of ALG-2 in membrane repair of metastazising cancer cells using a transwell chamber assay. Using this cellular system we wish to explore whether it is possible to further accelerate cancer cell membrane damage-induced death by interfering with ALG-2-dependent repair pathways. Understanding molecular mechanisms of membrane repair processes may help to develop novel cancer diagnostic/therapeutic approaches.

Methods:

            mammalian cell culturing

            Cell damaging assay - viability measurement

            Cell invasion experiments - transwell chamber assay

            FACS analysis

            microscopy

            ectopic gene expression

            molecular cloning

            Western blotting, immunoprecipitation

References:

  1. Jimenez AJ, Perez F. Plasma membrane repair: the adaptable cell life-insurance, Curr Opin Cell Biol, 2017, 47:  99
  2. Scheffer LL, Sreetama SC, Sharma N, Medikayala S, Brown KJ, Defour A, Jaiswal JK. Mechanism of Ca²⁺-triggered ESCRT assembly and regulation of cell membrane repair. Nat Commun., 2014, 5: 5646
  3. Berchtold MW. et al., ALG-2 participates in recovery of cells after plasma membrane damage by electroporation and digitonin treatment, PLoS One, 2018, 13: e0204520,
 
Anvendte metoder:cell cultivation (DT40, HELA), electroporation, viability assays, ectopic gene expression, calcium measurement, Western blotting, uptake of fluorescently labeled dextran sulfate
 
Keywords:membrane repair, Calcium binding proteins, cancer, ALG-2, ESCRT complex
 
Vejleder(e): Martin W. Berchtold
 
E-mail:mabe@bio.ku.dk