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A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast
1Division of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California School of Medicine, La Jolla 92093-0668.
Abstract:
We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4-64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4-64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4-64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4-64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.
Insights
FM 4-64 is a vital dye that stains yeast vacuole membranes and tracks endocytic transport. It acts as a marker for endocytic intermediates and reveals endosome-to-vacuole transport dynamics.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The vacuole is a crucial organelle in yeast, involved in various cellular processes.
- Understanding vacuolar transport and dynamics is essential for cell biology research.
- Existing vital stains may have limitations in tracking specific membrane trafficking events.
Purpose of the Study:
- To introduce and characterize FM 4-64 as a novel vital stain for yeast vacuole membranes.
- To utilize FM 4-64 to investigate bulk membrane internalization and transport to the vacuole.
- To explore the utility of FM 4-64 in studying vacuolar dynamics and endocytic pathways.
Main Methods:
- Employing the lipophilic styryl dye FM 4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide) as a vital stain in yeast.
- Observing FM 4-64 staining patterns at different temperatures (0°C, 15°C, 25°C, 30°C) to differentiate membrane localization.
- Analyzing FM 4-64 uptake and transport in various yeast mutants, including vacuolar protein sorting (vps) mutants and secretion (sec) mutants.
- Reconstituting FM 4-64 transport in permeabilized yeast spheroplasts to assess energy dependence.
Main Results:
- FM 4-64 specifically stains the vacuole membrane under steady-state conditions and acts as a vital stain for vacuolar membrane dynamics.
- FM 4-64 uptake is temperature-dependent, initially labeling the plasma membrane at low temperatures and subsequently trafficking to the vacuole.
- The dye effectively visualizes endocytic intermediates and endosome-to-vacuole transport, with staining patterns differing in specific vps mutants.
- FM 4-64 transport from endosome-like intermediates to the vacuole in vitro is dependent on ATP and cytosol.
Conclusions:
- FM 4-64 is a valuable new vital stain for the yeast vacuolar membrane.
- FM 4-64 serves as a reliable marker for endocytic intermediates and endosome-to-vacuole membrane transport.
- The dye provides insights into vacuolar dynamics, including events during mitosis and fusion/fission processes.