Confocal microscopy of FM4‐64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

  title={Confocal microscopy of FM4‐64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae},
  author={Sabine Fischer-Parton and Richard M. Parton and Patrick C. Hickey and Jan Dijksterhuis and Helen A. Atkinson and Nick D Read},
  journal={Journal of Microscopy},
Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4‐64, FM1‐43 or TMA‐DPH, three of the most commonly used membrane‐selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4‐64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its… 

Imaging vesicle trafficking and organelle dynamics in living fungal hyphae

The aims of the research were to develop and apply live cell imaging techniques using confocal microscopy to image and analyse vesicle trafficking, organelle dynamics and molecular localization at

FRET analysis of transmembrane flipping of FM4–64 in plant cells: is FM4–64 a robust marker for endocytosis?

It is demonstrated that under conditions that do not severely compromise cell viability, the FM4–64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP.

FM‐dyes as experimental probes for dissecting vesicle trafficking in living plant cells

Background information on the important characteristics of the FM‐dyes, and of optimal dye concentrations, conditions of dye storage, and staining and imaging protocols, are provided and particular emphasis is placed on using the FM-dyes in double labelling experiments to identity specific organelles.

Microinjecting FM4–64 validates it as a marker of the endocytic pathway in plants

It is concluded that FM4–64 is a specific marker for the endocytic pathway because the dye is pH‐sensitive, and the fluorescence intensity between the plasma membrane and tonoplast varied.

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea.

Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K-channel KAT1.

The data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K(+)-channel KAT1 from the PM via endocytic vesicles through the fate of fluorescent styryl dyes.

Uptake of the fluorescent probe FM4-64 by hyphae and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana.

Results suggest active uptake by different developmental stages of B. bassiana, including haemolymph-derived cells that can evade the insect immune system.

Endocytosis and vesicle trafficking during tip growth of root hairs

The localization of endocytosis in the tip is shown and specific endomembrane compartments and their recycling are indicated to show the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material.

Clathrin localization and dynamics in Aspergillus nidulans

Tests of FM4‐64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans, and it was found that ClaH did not colocalize well with the endocytic patch marker fimbrin.



Endocytosis and membrane turnover in the germ tube of uromyces fabae

It is concluded from measurements of membrane fluorescence that the turnover time from endocytosis to secretion of the dye amounts to 15 min, the first evidence for endocyTosis in a fungal germ tube.

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

It is shown 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.


Investigation of the distribution of the actin cytoskeleton in hyphal tips of the plant pathogenic fungus Sclerotium rolfsii suggested that the fibrillar coating of filasome contained actin and that filasomes represented the ultrastructural equivalent of actin plaques.

Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkörper, cytoskeleton and endomembranes after freeze-substitution.

The ultrastructure of freeze-substituted tip cells of Fusarium acuminatum was analysed and it is suggested that cytoskeletal elements play important roles in localized cell wall formation and the filasome, a previously unreported type of coated vesicle in fungi, might be involved in wall synthesis.

Yeast endocytosis assays.

Morphology of the yeast endocytic pathway.

Experiments examining endocytosis in the sec18 mutant showed an accumulation of positively charged Nanogold in approximately 30-50 nm diameter vesicles, lending strong support to the idea that the internalization step of endocyTosis in yeast involves formation of small vesicle of uniform size from the plasma membrane.

Imaging exocytosis and endocytosis

The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells

From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model.

Structure, function, and motility of vacuoles in filamentous fungi

Evidence is provided for a role of the tubular vacuoles in longitudinal transport of P. tinctorius cells and for the styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, which show little or no labeling of internal membranes in undamaged P. tournais cells.

Endocytosis and molecular sorting.

  • I. Mellman
  • Biology, Chemistry
    Annual review of cell and developmental biology
  • 1996
This review attempts to integrate emerging concepts concerning the protein-based signals responsible for molecular sorting and the cytosolic complexes responsible for the decoding of these signals to present a more coherent picture of how the endocytic pathway is organized and how the intracellular transport of internalized membrane components is controlled.