INVESTIGADORES
AGUILAR Pablo S.
congresos y reuniones científicas
Título:
Flow cytometry-based cell-cell fusion assay
Autor/es:
VALENTINA SALZMAN; VALENTINA PORRO; MARIELA BOLLATI; PABLO AGUILAR
Reunión:
Workshop; Cell-cell fusion EMBO Workshop; 2013
Resumen:
<!--
/* Font Definitions */
@font-face
{font-family:"Cambria Math";
panose-1:2 4 5 3 5 4 6 3 2 4;
mso-font-charset:0;
mso-generic-font-family:auto;
mso-font-pitch:variable;
mso-font-signature:-536870145 1107305727 0 0 415 0;}
@font-face
{font-family:Calibri;
panose-1:2 15 5 2 2 2 4 3 2 4;
mso-font-charset:0;
mso-generic-font-family:auto;
mso-font-pitch:variable;
mso-font-signature:-520092929 1073786111 9 0 415 0;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal
{mso-style-unhide:no;
mso-style-qformat:yes;
mso-style-parent:"";
margin-top:0in;
margin-right:0in;
margin-bottom:10.0pt;
margin-left:0in;
line-height:115%;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:Calibri;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-fareast-font-family:Calibri;
mso-fareast-theme-font:minor-latin;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-bidi-font-family:"Times New Roman";
mso-bidi-theme-font:minor-bidi;
mso-ansi-language:ES;}
.MsoChpDefault
{mso-style-type:export-only;
mso-default-props:yes;
font-size:11.0pt;
mso-ansi-font-size:11.0pt;
mso-bidi-font-size:11.0pt;
font-family:Calibri;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-fareast-font-family:Calibri;
mso-fareast-theme-font:minor-latin;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-bidi-font-family:"Times New Roman";
mso-bidi-theme-font:minor-bidi;
mso-ansi-language:ES;}
.MsoPapDefault
{mso-style-type:export-only;
margin-bottom:10.0pt;
line-height:115%;}
@page WordSection1
{size:595.3pt 841.9pt;
margin:70.85pt 85.05pt 70.85pt 85.05pt;
mso-header-margin:35.4pt;
mso-footer-margin:35.4pt;
mso-paper-source:0;}
div.WordSection1
{page:WordSection1;}
-->
The fusion of haploid yeast cells of opposite mating types provides a
genetically tractable model system to study cell-cell fusion. Sacharomyces cereviseae mates
spontaneously and forms stable diploids. Genetic analysis has identified a
significant number of important players of the fusion process, nevertheless the
key components have not yet been found. Reports of cell fusion efficiency are
based on fluorescence microscopy assays of mating pairs, with one partner
expressing a soluble cytosolic fluorescence protein. Nonproductive mating pairs
that fail to fuse can either lyse or remain unfused with their plasma membranes
in close apposition. Although this method has been extensively used it is
time-consuming, limiting both the number of cell-cell fusion events and strains
that can be analyzed at the same time. We therefore have developed a new flow
cytometry-based cell-cell fusion assay. A defect in cell-cell fusion can be
measured easily with this technique and the three outcomes of mating can be
clearly differentiated: fused, non fused and lysed pairs. For this purpose,
MATa and MATalpha fluorescent ConA labeled haploid cells are co-incubated to
allow zygote formation. Each strain synthesizes a fluorescent protein fragment
that will form a bright fluorophore only by bi-molecular complementation. Thus,
in this assay the accumulation of four populations of fluorescent cells is
scored: triple-positive (fused pairs), double-positive (non fused pairs) and
simple-positive (haploid a or haploid alpha). A modification of the protocol
enables quantification of lysis as well. Wild type and deletions strains in
fusion related genes where analyzed using fluorescence microscopy as the
reference technique. Results obtained with both methods clearly reproduced
published data. With this new method thousands of mating pairs can be analyzed
every second providing quantitative, accurate and reproducible data allowing
for the first time to quickly quantified cell fusion efficiency in yeast.
