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Tracing the endocytic pathway of Aspergillus nidulans with FM4-64
1Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain. penalva@cib.csic.es
Abstract:
Simple procedures using FM4-64 to follow membrane internalization and transport to the vacuolar system and endomembranes in Aspergillus nidulans are described. FM4-64 internalization is energy, temperature and F-actin dependent, strongly suggesting that it occurs by endocytosis. The dye sequentially labels: (i) cortical punctuate organelles whose motility resembles that of yeast actin patches; (ii) approximately 0.7 microm circular, hollow structures representing mature endosome/vacuole; and (iii) intermediate and large (2-3 microm in diameter) size vacuoles whose lumen is strongly labeled with 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). These large vacuoles possibly correspond to the final stage of one branch of the endocytic pathway. In addition, FM4-64 labels strongly the mitochondrial network and weakly the nuclear membrane. A class of cytoplasmic punctuate organelles which become fluorescent very shortly after dye loading and that can move in either apical or basal direction at an average rate of 2-3 microm s(-1) is also described. This work provides a useful framework for the phenotypic characterization of A. nidulans mutants affected in endocytosis.
Insights
This study details FM4-64 dye procedures to track endocytosis in Aspergillus nidulans, revealing energy-dependent membrane transport to the vacuolar system and endomembranes.
Area of Science:
- Cell Biology
- Mycology
- Molecular Biology
Background:
- Endocytosis is a crucial cellular process for nutrient uptake and membrane trafficking.
- Understanding endocytic pathways in filamentous fungi like Aspergillus nidulans is vital for cell biology research.
Purpose of the Study:
- To describe simple FM4-64 staining procedures for visualizing endocytosis in Aspergillus nidulans.
- To characterize the dynamics and destinations of FM4-64 labeled structures within Aspergillus nidulans cells.
Main Methods:
- Utilized FM4-64 fluorescent dye to track membrane internalization and transport.
- Observed dye-dependent labeling of organelles including cortical punctate structures, endosomes, and vacuoles.
- Employed 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA) for lumenal labeling of vacuoles.
Main Results:
- FM4-64 internalization was found to be energy, temperature, and F-actin dependent, indicating endocytosis.
- Sequential labeling of cortical punctate organelles, mature endosomes/vacuoles, and larger vacuoles was observed.
- Mitochondrial networks and nuclear membranes were also labeled by FM4-64, alongside motile cytoplasmic organelles.
Conclusions:
- The described FM4-64 procedures provide a robust framework for studying endocytosis in Aspergillus nidulans.
- This method facilitates the phenotypic characterization of Aspergillus nidulans mutants affecting endocytic processes.
- The study elucidates key stages and components of the endocytic pathway in this fungal model organism.
