A model for the germ tube formation and mycelial growth form of Candida albicans

A model based on morphological and ultrastructural evidence is presented which illustrates a novel and hitherto undescribed pattern of germ tube formation and hyphal growth in early and mature colonies of Candida albicans. Accordingly, most of the cytoplasm within the parent yeast cell migrates into and forward with the extending germ tubes and leaves behind an extensively vacuolated yeast cell. Growing hyphae similarly are subtended by migrating "slugs' of protoplasm and leave behind vacuolated intercalary compartments. The vacuolated cell compartments apparently must first regenerate their protoplasmic contents before producing branches or secondary germ tubes. This model is used to explain certain unusual features of the growth kinetics of the filamentous form of this organism.     

Cytological interrelationships between the cell cycle and duplication cycle of Candida albicans

The cytology of nuclear division and septation in the yeast and hyphal phases of Candida albicans growing at 37 degrees C has been studied by fluorescence microscopy after staining of specimens with 4'6-diaminido-2-phenylindole (DAPI) and Calcofluor. Yeast and hyphal cells replicated their nuclei at about 18 min after the emergence of a bud or germ-tube. The site of nuclear division coincided with the future location of the septum in both forms. This occurred at the junction of the bud and parent yeast cell or 6.0 micron from the parent yeast in germ tubes which were formed in medium containing serum. The filamentous forms of a range of clinical and laboratory strains grown in a variety of germ tube-inducing media were all extensively vacuolated. Germ tube extension in all of these media was linear. It is suggested that there is little biosynthesis of cytoplasm during the initial stages of germ tube growth in this organism and that this accounts for the development of the large vacuoles and the linear growth kinetics.     

Fine Structure of Quiescent and Germinating Aeciospores of Cronartium fusiforme

Aeciospores of the long-cycle heteroecious rust fungus, Cronartium fusiforme, were found to have an extremely thick cell wall with striking spicules protruding from it. The wall was readily degraded by commercial chitinase, but spicules were unaffected. Quiescent spores contained two nuclei with distinct nuclear membranes possessing many pores. Numerous membrane-bounded lipid bodies were found both in wild-type orange and in white mutant aeciospores. An abundance of irregularly ovoid mitochondria was present in quiescent spores. After glutaraldehydeosmium fixation, the surface of the mitochondria appeared to be covered with ribosomes or microtubules in a paracrystalline array, whereas after permanganate fixation only smooth outer mitochondrial membranes were noted. The latter fixative revealed abundant vesicular endoplasmic reticulum in the spore. Spores incubated at 20 C on agar produced one to five distinct germ tubes within 65 to 180 min. These thin-walled tubes exhibited varying degrees of branching, and reached a total hyphal length of 300 to 500 mu prior to rupturing. Emergence of germ tubes took place through a pore in the spore wall and appeared to be mainly a physical flowing of cytoplasm from the spore into the germ tube without division of nuclei or other cell organelles. On completion of germination, the protoplasm of the germ tube contained both nuclei and nearly all of the other spore contents. Mitochondria had smooth outer membranes, were greatly elongated, and possessed distinct longitudinal cristae. A limited amount of rough endoplasmic reticulum was arranged parallel to the germ tube wall. Other organelles seen in germ tubes were lipid bodies, concentric membrane figures, and numerous ribosomes. Lipid bodies appeared smaller and fewer in number than in quiescent spores.     

A Candida albicans Temperature-Sensitive cdc12-6 Mutant Identifies Roles for Septins in Selection of Sites of Germ Tube Formation and Hyphal Morphogenesis

Septins were identified for their role in septation in Saccharomyces cerevisiae and were subsequently implicated in other morphogenic processes. To study septins in Candida albicans hyphal morphogenesis, a temperature-sensitive mutation was created that altered the C terminus of the essential Cdc12 septin. The cdc12-6 cells grew well at room temperature, but at 37°C they displayed expected defects in septation, nuclear localization, and bud morphogenesis. Although serum stimulated the cdc12-6 cells at 37°C to form germ tube outgrowths, the mutant could not maintain polarized hyphal growth and instead formed chains of elongated cell compartments. Serum also stimulated the cdc12-6 mutant to induce a hyphal reporter gene (HWP1-GFP) and a characteristic zone of filipin staining at the leading edge of growth. Interestingly, cdc12-6 cells shifted to 37°C in the absence of serum gradually displayed enriched filipin staining at the tip, which may be due to the altered cell cycle regulation. A striking difference from the wild type was that the cdc12-6 cells frequently formed a second germ tube in close proximity to the first. The mutant cells also failed to form the diffuse band of septins at the base of germ tubes and hyphae, indicating that this septin band plays a role in preventing proximal formation of germ tubes in a manner analogous to bud site selection. These studies demonstrate that not only are septins important for cytokinesis, but they also promote polarized morphogenesis and selection of germ tube sites that may help disseminate an infection in host tissues.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidial germination and outgrowth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of conidia was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged conidia to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiogenesis. An ultrastructural study of the germination stage indicated nuclear migration into the emerging germ tube whose new cell wall was an extension of the parent conidium's innermost cell wall layer. Septal formation at the neck of the germ tube followed. The septal pore was filled with particulate material and the septal membranes possessed unusual linear elements in their median hydrophobic zones. The germ tube, which possessed a smooth-surfaced plasma membrane, continued to elongate with periodic septum formation. The parent conidium and later the proximal germ tube showed progressive vacuolation and the cytoplasm became largely occupied by electron-translucent material. In older cells the septal pore was blocked by Woronin bodies. Compared with normal conidial germination this microcycle' germination is far more synchronous and the resultant germling is morphologically simpler. In ultrastructural terms, however, germination appears to be identical with that obtained at 28 degrees C.     

Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae

Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.     

Role of the conidium in dimorphism of Blastomyces dermatitidis

Fine details of yeastlike cell development of Blastomyces dermatitidis from its conidium are described and illustrated by electron micrographs. When cultured in an enriched medium at 37 °C, conidia of two strains of B. dermatitidis readily underwent ultrastructural changes consistent with mycelial to yeast dimorphism. Although hyphal cells contained in the conversion cultures were observed consistently to undergo profound degenerative changes, the conidia rapidly germinated to give rise to short germ tubes which subsequently enlarged to form intermediate yeast mother cells (YMC). The wall of the germ tube arose from the innermost layer of the wall of the germinant. During the transition globoid osmiophilic inclusions of unknown origin and function were observed in vacuolated areas of the germ tube and YMC cytoplasm. Yeastlike daughter cells then budded from the intermediate YMC. Since transformation was readily accomplished under in vitro conditions favoring mycelial to yeast dimorphism, it is suggested that the conidium of B. dermatitidis represents the primary infective unit of this pathogenic fungus.     

Influence of applied electrical fields on yeast and hyphal growth of Candida albicans

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.     

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi

Summary. The membrane-selective fluorescent dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide, was used to stain the apical vesicle cluster within the specialized Spitzenkörper of the germ tube of the rust fungi Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. The Spitzenkörper stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spitzenkörper was asymmetrically positioned close to the cell–substratum interface during germ tube growth. The Spitzenkörper showed variations in shape and positioning over short (5 s) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spitzenkörper supplies secretory vesicles for germ tube growth. A pronounced Spitzenkörper disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spitzenkörper, was again observed during the formation of the highly polarized penetration peg.Correspondence and reprints: Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.Received October 25, 2002; accepted February 26, 2003; published online August 26, 2003     

Molecular and cellular events during the yeast to mycelium transition in Sporothrix schenckii

Unbudded singlets from exponentially growing yeast cells of Sporothrix schenckii were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 degrees C. These conditions supported only the development of the mycelial form of S. schenckii in a reproducible manner which allowed further analysis of the early cellular events occurring during the yeast-to-mycelium transition. The relationship between macromolecular synthesis (DNA and RNA synthesis) and nuclear division, hyphal growth and septum formation were investigated during germ tube formation. RNA synthesis started 0 to 3 h after the induction of germ tube formation, followed by DNA synthesis and the first nuclear division, which took place between 3 and 6 h. Germ tube formation followed nuclear division and was first evidenced 6 h after the induction of germ tube formation, but was not completed until 12 h after inoculation. Septation was first observed in these germ tubes at the mother cell-germ tube junction 6 h after induction. Addition of hydroxyurea, an inhibitor of DNA synthesis, to the medium, also inhibited nuclear division and germ tube growth, suggesting that these processes in S. schenckii are dependent upon DNA synthesis.     

The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins.

A hyphally regulated gene (HYR1) from the dimorphic human pathogenic fungus Candida albicans was isolated and characterized. Northern (RNA) analyses showed that the HYR1 mRNA was induced specifically in response to hyphal development when morphogenesis was stimulated by serum addition and temperature elevation, increases in both culture pH and temperature, or N-acetylglucosamine addition. The HYR1 gene sequence revealed a 937-codon open reading frame capable of encoding a protein with an N-terminal signal sequence, a C-terminal glycosylphosphatidylinositol-anchoring domain, 17 potential N glycosylation sites, and a large domain rich in serine and threonine (51% of 230 residues). These features are observed in many yeast cell wall proteins, but no homologs are present in the databases. In addition, Hyr1p contained a second domain rich in glycine, serine, and asparagine (79% of 239 residues). The HYR1 locus in C. albicans CAI4 was disrupted by "Ura-blasting," but the resulting homozygous delta hyr1/delta hyr1 null mutant displayed no obvious morphological phenotype. The growth rates for yeast cells and hyphae and the kinetics of germ tube formation in the null mutant were unaffected. Aberrant expression of HYR1 in yeast cells, when an ADH1-HYR1 fusion was used, did not stimulate hyphal formation in C. albicans or pseudohyphal growth in Saccharomyces cerevisiae. HYR1 appears to encode a nonessential component of the hyphal cell wall.     

Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores

Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.     

Formation and regeneration of protoplasts of Sclerotium glucanicum

Summary Protoplast yields from Sclerotium glucanicum using Novozym 234 as the lytic enzyme were affected by the osmotic stabilizers selected, the incubation conditions used for wall degradation, and culture age. Scanning electron microscopic observations revealed that protoplast release from all hyphal regions gradually followed random wall attack, and nuclear staining showed that some protoplasts contained as many as eight nuclei. Their regeneration involved germ tube production on solid media, but formation of chains of buds and possibly cytoplasmic cleavage in liquid medium. Regenerated protoplasts gave similar exopolysaccharide yields to those of the parent culture.     

Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.     

Do single mitochondria contain zones with different membrane potential?

Observations of Lan Bo Chen’s group using a mitochondria-selective fluorochrome 5,5’,6,6’- tetrachloro- 1,1’,3,3’- tetraethylbenzimidazolocarbocyanine iodide (JC-1) indicate that mitochondria in situ may have zones of different electrochemical potential along their length. This was indicated by the formation of J-aggregates of this dye at distinct sites along a single mitochondrion. Also, intensity variations along single mitochondria were found with diamino-styryl-pyridinium methiodide (DASPMI), another fluorochrome that selectively stains mitochondria depending on their electrochemical potential. DASPMI exchanges easily with the cytoplasm and changes its quantum yield when bound to mitochondrial membranes. Therefore, fluorescence intensity is primarily controlled by the membrane environment rather than by mass accumulation. Two possible explanations of intramitochondrial fluorescence intensity variations have to be discussed: variations in the amount of mitochondrial inner membrane per unit of projection area (or voxel), and differences in the electrochemical gradient. This problem has been approached by comparing fluoro-micrographs of mitochondria in endothelial cells stained with either JC-1 or DASPMI with electron micrographs of the same mitochondria after fixation with glutardialdehyde and osmium tetroxide and ultrathin sectioning. JC-1 red fluorescence (revealing J-aggregate formation) as well as high-intensity staining with DASPMI correlate roughly with the local thickness of mitochondria; no differences in the crista organization are revealed for those areas or mitochondria exhibiting red JC-1 fluorescence and those with green fluorescence. The distance between red fluorescing areas in a single mitochondrion seem to be caused by competition for dye molecules placed in between centres of JC-1 aggregation. Isolated mitochondria are of uniform small size and spherical shape; therefore, no differences in shape interfere with JC-1 staining. Thus JC-1 may be an appropriate indicator of membrane potential in isolated mitochondria. In living cells mitochondria often are large and elongated, and thus the situation is not straightforward to interpret. However, evidence is provided that there are submitochondrial zones, which differ in membrane potential from one adjacent area to another, because DASPMI staining of intramitochondrial zones reveals differences in fluorescence intensity and preferred photodamage of these areas. In some cases separation of the zones of higher membrane potential by cristae traversing the whole diameter of a mitochondrion has been observed. Local photobleaching of stained mitochondria results in a loss of fluorescence along the total length of a mitochondrion. However, this type of bleaching develops over tens of seconds, not in the very short time range (e.g. ms) expected from the discharge of all the membranes if they were electrically coupled.     

Ultrastructure of germination and mucilage production inHalosphaeria appendiculata (Halosphaeriaceae)

The germination of ascospores of the marine fungusHalosphaeria appendiculata was investigated with transmission electron microscopy. Prior to germination, settled ascospores became surrounded by a fibro-granular layer. Small, membrane-bounded vesicles and larger electron-dense membrane-bounded vesicles aggregated at the site of germ tube formation where the plasmalemma adjacent to the aggregation was convoluted. The vesicles appeared to fuse with the plasmalemma, releasing their contents. Enzymatic digestion of the spore wall probably occurred at the time of germ tube emergence. After the nucleus had migrated into the newly formed germ tube, a septum was formed to delimit the germ tube from the ascospore. The growing germ tube can be divided into 3 morphological regions, namely the apical, sub-apical and vacuolated regions, and is typical of other fungi. A mucilaginous sheath was associated with the older mycelium. The germ tube displaced the polar appendage, and the ascospore, germ tube and appendage were enclosed in a mucilaginous sheath. In ascospores which subtended old germ tubes, the nucleus and lipid body became irregular in shape and the cytoplasm was more vacuolated. Microbody-like structures remained associated with the lipid throughout development, and were present in old ascospores.     

Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogen Candida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) (), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth in S. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother-daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.     

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.     

Morphological studies of N-acetylglucosamine induced germ tube formation by Candida albicans

In N-acetylglucosamine induced germ tube formation by Candida albicans, multiple (up to five) protuberances appeared within 90 min at 37 degrees C on each yeast cell. The protuberances were extensions of the cytosol and contained vesiclelike structures. Usually only one protuberance subsequently developed into a germ tube. The germ tubes emanated from all aspects of the cell surface but seldom from the budding (long axis) poles. Pseudohyphae, which originated from the budding pole, exhibited a marked constriction at the site of emergence and were 0.6-2.5 microns in diameter compared with a diameter of 0.6-0.8 micron for germ tubes. The presence of septa confirmed that germ tubes are precursors of septate mycelia. Ultrathin-section transmission electron microscopy of aldehyde plus osmium fixed cells revealed electron-lucent walls with a thin electron-dense outer layer. A fibrillar border was also routinely associated with germ tubes. Poststaining with potassium permanganate revealed, in addition, a previously invisible fuzzy layer on the outer region of the cell wall which extended over bud scars and germ tubes and which coalesced at sites of contact between cells.     

How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells

Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (τ₁) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions τ₂ and τ₃ increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of π-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.