Technical Reference #1829

1829.

Myosin 1G Is an Abundant Class I Myosin in Lymphocytes Whose Localization at the Plasma Membrane Depends on Its Ancient Divergent Pleckstrin Homology (PH) Domain (Myo1PH) Genaro Patino-Lopez; L. Aravind; Xiaoyun Dong; Michael J. Kruhlak; E. Michael Ostap; and Stephen Shaw, National Institutes of Health - NCI, Journal of Biological Chemistry, 285(1829), (2010)
Link To Paper

Keywords:
Cytoskeleton; Evolution; Lymphocyte; Myosin; Plasma Membrane; Protein Domains; Class I Myosin; PH Domain

Materials & Methods:
Immunofluorescence Microscopy—PBT Jurkat and 300.19 cells (1–2 106 cells each) were dropped onto the glass bottom of 35-mm MatTek culture dishes (MatTek Ashland MA) precoated with poly-L-lysine (Sigma). Cells were allowed to settle for 10 min at room temperature and either analyzed alive or fixed (for endogenous Myo1G expression and 5-Ptase recruitment to the membrane) by the addition of 4% paraformaldehyde solution (Sigma). After 10 min at room temperature samples were washed with PBS permeabilized with 0.2% Triton X-100 and blocked for 1 h at room temperature in PBS with 3% BSA. Primary antibodies were added for 1 h at room temperature in PBS with 3% BSA. After washing goat anti-rabbit Alexa488-conjugated secondary antibodies (Molecular Probes) in PBS with 3% BSA were added for 1 h at room temperature. Samples were examined using a Zeiss LSM510 laser scanning confocal microscope using a 63 or 100 objectives (Carl Zeiss). Quantitative analysis was performed using the Imaging Examiner software (LSM Carl Zeiss Inc.). For each cell analyzed a line was drawn manually at the plasma membrane and another line was drawn just inside the plasma membrane in the cytosol. Average fluorescence intensity was determined for the set of pixels touching the plasma membrane line (plasma membrane mean intensity) and for those touching the cytosol line (cytosol mean intensity). Membrane enrichment for that cell is calculated as the (plasma membrane mean intensity)/(cytosol mean intensity). Results are summarized as the mean S.E. from at least eight representative cells from two to three independent experiments. Because pixels touching the line at the plasma membrane will often include a fraction of extracellular space the average obtained for a construct that is not enriched at the plasma membrane is often somewhat less than 1.0.

Microscopic Technique
Immunofluorescence Microscopy

Cell Type(s)
PBT; Jurkat; and 300.19 cells