Fine Structure of Hepatozoon mocassini (Apicomplexa,Eucoccidiorida) Gamonts and Modifications of the Infected Erythrocyte Plasmalemma1

Transmission electron microscopy and scanning electron microscopy were used to investigate the fine structure of Hepatozoon mocassini gamonts and modifications of the infected erythrocyte plasmalemma. Intraerythrocytic gamonts were contained within a parasitophorous vacuole. An electron-lucid space observed between the gamont pellicle and the membrane of the vacuole corresponded to the unstained space described in light microscopy studies. Gamonts possessed a conoid, polar ring, subpellicular microtubules, four pairs of rhoptries, micronemes, ovoid granules, mitochondria with tubular cristae, and a pellicle composed of three individual unit membranes. The conoid had an anterior diameter of 320 nm, a posterior diameter of 360 nm, and a length of 150 nm. In contrast to a report on Hepatozoon aegypti, no micropore or “canopy-like structure” was observed. The plasmalemma of infected erythrocytes exhibited two types of modifications: gross membrane deformations and knobs with an electron-dense central mass. These knobs are structurally distinct from previously described membrane excrescences.     

The Fine Structure of the Colonial Kinetoplastid Flagellate Cephalothamnium cyclopum Stein

Cell structure, cell adhesion, and stalk formation have been examined by electron microscopy in the colonial flagellate, Cephalothamnium cyclopum. Each cell is obconical or spindle-shaped, pointed posteriorly and truncated anteriorly. The cell membrane is underlain by epiplasm 0.1 μm thick in the posterior region, but bands of microtubules support the anterior region which is differentiated into a flagellar pocket, oral apparatus and contractile vacuole. Each of 2 flagella, joined a short way above their bases by an interflagellar connective, has a paraxial rod and mastigonemes. One flagellum is free and is important in food gathering while the other is recurrent and lies in a shallow groove on the ventral cell surface but projects posteriorly into the stalk. The basal bodies of these flagella are bipartite structures connected by a pair of striated rootlets with accessory microtubular fibers. The oral apparatus consists of a funnel-shaped buccal cavity and cytostome. It is supported by helical and longitudinal microtubules and also has nearby striated and microtubular fibers. Possible roles of associated oral vesicles in relation to ingestion are discussed. A reticulate mitochondrion houses a massive kinetoplast which has a fibrillar substructure resembling that of dinoflagellate chromosomes. Adjacent flagellates adhere by laminate extensions of their posterior regions and attach by their recurrent flagella to a communally secreted stalk composed of finely fibrillar material. This study indicates that Cephalothamnium belongs in the order Kinetoplastida, and has many features in common with members of the family Bodonidae.     

DEVELOPMENT OF PERIPHERAL VACUOLES IN PLANT CELLS

The vacuolar apparatus of various plant cells consists of two distinct features: the large central vacuole and peripheral vacuoles which are derived from invaginations of the plasma membrane. Peripheral vacuoles are conspicuous structures in both living and fixed hair or filament cells of Tradescantia virginiana. They occur as spherical structures along the inner boundary of the peripheral cytoplasm and can be recognized as projections into the central vacuole. These structures are variable in size and number within a cell and can represent a significant proportion of the volume of the vacuole. Peripheral vacuoles most frequently are observed in motion with the streaming cytoplasm although their velocity is usually somewhat slower that that of the cytoplasmic organelles. Ultrastructural studies show two closely approximated membranes, one for each vacuole, in areas where a peripheral vacuole projects into the central vacuole. These are separated by an intermembrane zone continuous with the peripheral cytoplasm. The movement of organelles over the perimeter of the peripheral vacuole is presumed to occur along this intermembrane zone. The internal area of the peripheral vacuoles may appear empty although some contain a vesicular content of unknown origin and function.     

The Mode of Function of the Cytopharyngeal Basket of the Ciliate Pseudomicrothorax dubius

The ciliate Pseudomicrothorax dubius feeds on filamentous blue-green algae, ingesting them at rates of up to 15 μm per second, by means of a cytopharyngeal basket. The wall of the basket is composed of 22 ± 3 nemadesmata, each of which is a bundle of about 200 microtubules which are cross-linked in a hexagonal pattern. The lumen of the non-feeding basket is filled with cytoplasma into which project the nemadesmal lamellae. Each nemadesmal lamella is attached to a nemadesm and consists of a single row of 20–30 microtubules. Each microtubule of the nemadesmal lamella bears a row of pairs of arm-like projections which are embedded in a filamentous matrix. During feeding, the lumen of the basket is occupied by the developing food vacuole. The nemadesmal lamellae are observed between the vacuole membrane and the nemadesmata, and the arms of the nemadesmal lamellae microtubules are oriented toward the membrane of the food vacuole or of small vesicles. A mechanism for the generation of force for phagocytosis by means of the microtubule arms is proposed.
During food uptake the membrane of the food vacuole increases rapidly at rates up to 270 μm² per second. Vacuole growth results from the fusion of membrane-bound vesicles. During phagocytosis a fast streaming of these vesicles can be observed in the cytoplasm surrounding the basket. The direction of streaming is opposite to that of ingestion of the algal filament. The vesicles enter the lumen of the basket at its anterior end, in a zone where the wall of the basket is perforated.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Fine-Structural Aspects of Macrogametogenesis in Eimeria magna*†

SYNOPSIS. Macrogamonts in tissues from rabbits killed 5 1/2 days after inoculation with Eimeria magna oocysts were studied with the electron microscope. In young macrogamonts, parts of cytoplasm, sometimes including micronemes, were pinched off into the parasitophorous vacuole. In all stages of development, small segments of the inner membrane complex were present beneath the limiting membrane. Micropores also were seen in all stages, and some apparently functional ones were present in mature macrogametes. Wall-forming bodies of Type I and Type II were observed in relatively early stages. The former were less numerous than the latter, which had a more compact appearance than in other species. Usually, several Golgi complexes were present and several Golgi adjuncts occurred in the vicinity of the nucleus in all stages of development. Microgametes were observed in the cytoplasm of host cells harboring immature macrogametes.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host‐derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC‐1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno‐EM revealed that the ATP‐delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria‐vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP‐delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.     

CELL FORM AND SURFACE PATTERNS IN CHROOMONAS and CRYPTOMONAS CELLS (CRYPTOPHYTA) AS REVEALED BY SCANNING ELECTRON MICROSCOPY1

Fifteen freshwater cryptomonad species were freeze-dried and examined with the scanning electron microscope. Surveys of cell surfaces revealed four general cell types. Chroomonas type cells lack a furrow but possess a shallow vestibular depression where the flagella are inserted. The presence of a gullet could not be detected. Cryptomonas spp. displayed three morphological types, all lacking gullets. The first type of Cryptomonas has a simple, shallow furrow with ridges that apparently can close to form a raphe but an oval opening or stoma remains at the posterior end and an opening from the vestibulum is formed at the anterior end. The second Cryptomonas type consists of a complex furrow with furrow ridges and folds that extend almost two-thirds of the cell length. A sloma is present in the central region of the closed furrow. The folds apparently can separate thereby exposing the underlying furrow. The third type of Cryptomonas possesses a simple, non-closing furrow. At the anterior end there is a vestibular ligule which extends from the dorsalleft side of the cell and covers the region of the vestibulum where the contractile vacuole discharges.     

Electron microscopy and image analysis of the mitochondrial outer membrane channel,VDAC

The channel protein in the outer membrane ofNeurospora crassa mitochondria, VDAC, forms extended planar crystals on the membrane. The arrays, which are induced by phospholipase A₂, are polymorphic, varying from parallelogram (P) to near-rectangular (R) geometry with increased phospholipase treatment. Computer-based analysis of projection images of negatively stained VDAC arrays indicates that the protein forms a transmembrane channel in the P array. Comparison of average images of arrays embedded in different negative stains suggests that the bore of the channel is 2–2.5 nm. The locations of functionally important lysine clusters on VDAC are inferred from the effects of succinylation on projection images of arrays negatively stained with phosphotungstate. Projection images of unstained frozen-hydrated arrays indicate the general shape of the channel and suggest each channel is formed by one 31-kDa VDAC polypeptide.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

Membrane Dynamics of the Contractile Vacuole Complex of Paramecium1

ABSTRACT. Membrane dynamics of the contractile vacuole complex of Paramecium were investigated using conventional electron microscopy of cells so that the vacuoles were serial-sectioned longitudinally and transversely. During systole, vacuolar membrane collapses first into flattened cisternae which undergo further modification into a mass of interconnected small membrane tubules. These tubules retain their connections with the radiating microtubular ribbons; consequently they are found only in the poleward hemisphere. Permanent connections between ampullae and the collapsed vacuole membrane could not be verified nor was a sphincter-like mechanism for closing such a junction observed. Membranes of the ampullae and the collecting canals also collapse to varying extents into arrays of tubules that remain bound to microtubular ribbons during diastole. Thus vacuole, ampullae, and collecting canal membranes all assume tubular forms when internal volume is at a minimum. Having failed to observe a microfilamentous encasement of the vacuole, we suggest that an alternative mechanism for the “contractile” function should be sought. One such is based on fluid volume increase and fluid flow within transiently interconnected tubular membrane systems that cycle between a tubular and a planar membrane form as internal volume is periodically increased and reduced. The driving force for this mechanism might best be sought in the molecular structure of the membranes of the contractile vacuole complex.     

Dual turgor regulation response to hypotonic stress in Lamprothamnium papulosum

Cells of the salt-tolerant charophyte Lamprothamnium respond differently to hypotonic challenge according to their position on the plant (i.e. cell age). Differences in electrophysiological response are coupled with differences in cell fine structure, and the presence or absence of extracellular mucilage. (1) Young, apical (fast-regulating, FR) cells respond with sudden cessation of cyclosis, depolarization to –50 mV (in some cells by more than 100 mV) and increase in membrane conductance by up to an order of magnitude. Intracellular [K⁺]_v, [Na⁺]_v and [Cl^–]_v decrease 1 h after hypotonic challenge. Patch-clamping cytoplasmic droplets reveals two types of K⁺ channel, 150 pS and 35 pS, and a small conductance Cl^– channel, 35 pS (conductances at estimated tonoplast resting potential between zero and 20 mV). Extracellular mucilage is thin (< 5 μm thick) or lacking, similar to freshwater Chara. Unlike freshwater charophytes these cells have a canalicular vacuolar system of large surface area and compartment the fluorochrome 6 carboxyfluorescein in the cytoplasm rather than the vacuolar system. (2) Older basal (slow-regulating, SR) cells do not cease streaming on hypotonic challenge and depolarize only slightly (by approximately 20 mV) with small or no change in membrane conductance. After 1 h the intracellular [K⁺]_v, [Na⁺]_v and [Cl^–]_v scarcely change. Patch-clamping cytoplasmic droplets reveals two types of K⁺ channel, medium conductance 90 pS and low conductance (as in FR cells). The large conductance K⁺ channel was not observed. The Cl^– channel was more active in SR cells. The cells were coated with extracellular mucilage more than 10 μm thick. In a similar manner to freshwater Chara, these cells compartment 6 carboxyfluorescein in a large central vacuole. In the older cells, making up the bulk of any given plant, the simultaneous development of extracellular mucilage and a large central vacuole which compartments 6 carboxyfluorescein is associated with a minimal electrophysiological response to hypotonic challenge. The significance of these findings for salt-tolerance is discussed.     

An electron microscopic study of Babesia microti invading erythrocytes

Summary Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.Supported by Grant AI 08989 from the U.S. Public Health Service. The excellent technical assistance of Ms. Renata Klatt is gratefully acknowledged     

The Sodium Channel Has Four Domains Surrounding a Central Pore

The voltage-gated sodium channel generates the action potential. This 300-kDa protein has four homologous regions, which are also homologous to the voltage-sensitive tetrameric potassium channel. We isolated sodium channels fromElectrophorus electricuselectroplax by detergent solubilization and immunoaffinity chromatography and studied their structure by electron microscopy of negatively stained specimens. Different projections were aligned, classified, and averaged. In side view, the channel protein exhibits the shape of a truncated cone, 14 nm in height. One end has a diameter of 12 nm and is asymmetric, while the other is more symmetric and has a diameter of 7–10 nm. In top views, the sodium channel appears to consist of four domains of different size and to have a stain-filled pore in the center.     

The cell wall porin of the gram-positive bacterium Nocardia asteroides forms cation-selective channels that exhibit asymmetric voltage dependence

Detergent-solubilized cell wall extracts of the gram-positive, strictly aerobic bacterium Nocardia asteroides contain channel-forming activity as judged from reconstitution experiments using lipid bilayer membranes. The cell wall porin was identified as a protein with an apparent molecular mass of about 84 kDa based on SDS-PAGE. The porin was purified to homogeneity using preparative SDS-PAGE. The 84-kDa protein was no longer observed after heating in SDS buffer. The presumed dissociation products were not observed on SDS-polyacrylamide gels. The cell wall porin increased the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures by the formation of cation-selective channels, which had an average single-channel conductance of 3.0 nS in 1 M KCl. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated negative point charge effects on the channel properties. The analysis of the concentration dependence of the single-channel conductance using the effect of negative charges on channel conductance suggested that the diameter of the cell wall channel is about 1.4 nm. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The cell wall channel switched into substates, when the cis side of the membrane, the side of the addition of the protein, had negative polarity. Positive potentials at the cis side had no influence on the conductance of the cell wall channel. Received: 23 September 1998 / Accepted: 9 December 1998     

Localization of Pyrophosphatase and V-ATPase in Chlamydomonas reinhardtii

Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V-ATPase activities. By immunogold labelling we have shown that H⁺-pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V-ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V-ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 ? 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,-head of V-ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate-sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely-packed 6 ? 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5- and 6-fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V₀ body of the V-ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function.     

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca²⁺ in the cytoplasm ([Ca²⁺]_c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca²⁺]_c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca²⁺]_c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca²⁺-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.     

The Supramolecular Organization of Red Algal Vacuole Membrane Visualized by Freeze-fracture

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost clearly and more frequently in the exoplasmic fracture(EF) face of the vacuole membrane of all red algae investigated.The tetrads lie individually within the vacuole membrane orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

Tracking the dynamic interplay between bacterial and host factors during pathogen‐induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen‐induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram‐negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin‐3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high‐content and high‐throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.