Volume 8 issue 6 June 2007
The BEACH protein LvsB is localized on lysosomes and postlysosomes and limits their fusion with early endosomes
E Kypri, C Schmauch, M Maniak and A De Lozanne

Supplemental Figure 1: The loss of LvsB leads to abnormal fusion of early endosomes with postlysosomes. Wild type (A) and LvsB null (B) cells expressing GFP-vacuolin B were labeled for 10 min with TRITC-Dextran, chased in buffer, for 20-30 minutes and imaged by fluorescence microscopy. The three wild type cells shown here do not have any colocalization of the two markers. In wild type cells the internalized dextran did not colocalize with GFP-vacuolin during the first 30 minutes of chase. This indicates that the newly internalized marker normally needs more than 30 minutes to reach the postlysosomal compartment. In contrast, the three LvsB null cells shown here illustrate colocalization of dextran and GFP-vacuolin in less than 30 minutes of chase. This mutant phenotype could be the result of abnormal fusion of newly internalized endosomes ith vacuolin-labeled postlysosomes. (See Figure 5 for quantification at different time points after chase). Bar, 5 µm.

Figure 1 (jpg)

Supplemental Figure 2: Fusion of early and late endosomal compartments is increased in LvsB null cells. Wild type and LvsB null cells were labeled with FITC-Dextran for 5 minutes and then chased in buffer for 30 minutes to allow the internalized marker to reach late compartments in the cells.  Cells were then labeled with a 5 minute pulse of TRITC-Dextran, washed and imaged by fluorescence microscopy within 10 minutes.  In wild type cells the two pulses of Dextran remain in separate compartments.  In contrast, the two pulses of Dextran rapidly merge in LvsB null cells (See Figure 6 for quantification).  Bar, 10 µm.

Figure 2 (jpg)