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Multiphoton microscopy (MPM) is a powerful imaging technology for brain research. The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels. It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases. However, there has been no comparison with near-infrared (NIR) emission. In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo, we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828 nm and 620 nm, respectively. Both experimental and simulation results show that as the imaging depth increases, NIR emission decays less than orange-red fluorescent emission. These results show that it is preferable to shift the emission wavelength to NIR to enable more efficient signal collection deep in the brain. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM) is a powerful imaging technology for brain research. The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels. It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases. However, there has been no comparison with near-infrared (NIR) emission. In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo, we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828[Formula: see text]nm and 620[Formula: see text]nm, respectively. Both experimental and simulation results show that as the imaging depth increases, NIR emission decays less than orange-red fluorescent emission. These results show that it is preferable to shift the emission wavelength to NIR to enable more efficient signal collection deep in the brain.

Hyphae are microscopic filaments that elongate and branch to create networks of interconnected tubes. Understanding how they work remains a formidable challenge in experimental mycology. Important advances in hyphal research in the 20th century came from electron microscopy, which revealed clusters of cytoplasmic vesicles in the cell apex, and biochemical studies that identified the cell wall materials that are assembled at the tip. Early genetic experiments on hyphae based on mutant analysis were disappointing and provided little information on the relationship between genotype and phenotype. Progress has come more recently, in the first decades of this century, by combining the techniques of molecular genetics with modern imaging methods. Live-cell imaging has allowed us to study the dynamics of cell components in strains of fungi engineered with plasmids encoding proteins fused to fluorescent probes. This technology has provided significant insights on the growth process and yet the fundamentals of hyphal growth remain elusive. • Research on hyphal growth has advanced through live-cell imaging • Tracking the movement of proteins attached to fluorescent reporters has become a particularly valuable experimental tool • Many fundamental questions about the growth of fungal hyphae remain unanswered [ABSTRACT FROM AUTHOR]

Filamentous fungi grow by adding cell wall and membrane exclusively at the apex of tubular structures called hyphae. Growth was previously believed to occur only through exocytosis at the Spitzenkörper, an organised body of secretory macro- and microvesicles found only in growing hyphae. More recent work has indicated that an area deemed the sub-apical collar is enriched for endocytosis and is also required for hyphal growth. It is now generally believed that polarity of filamentous fungi is achieved through the balancing of the processes of endocytosis and exocytosis at these two areas. This review is an update on the current progress and understanding surrounding the occurrence of endocytosis and its spatial regulation as they pertain to growth and pathogenicity in filamentous fungi.

We investigated the mechanisms and the spatio-temporal dynamics of fluid-phase and membrane internalization in the green alga Chara australis using fluorescent hydrazides markers alone, or in conjunction with styryl dyes. Using live-cell imaging, immunofluorescence and inhibitor studies we revealed that both fluid-phase and membrane dyes were actively taken up into the cytoplasm by clathrin-mediated endocytosis and stained various classes of endosomes including brefeldin A- and wortmannin-sensitive organelles (trans-Golgi network and multivesicular bodies). Uptake of fluorescent hydrazides was poorly sensitive to cytochalasin D, suggesting that actin plays a minor role in constitutive endocytosis in Chara internodal cells. Sequential pulse-labelling experiments revealed novel aspects of the temporal progression of endosomes in Chara internodal cells. The internalized fluid-phase marker distributed to early compartments within 10 min from dye exposure and after about 30 min, it was found almost exclusively in late endocytic compartments. Notably, fluid cargo consecutively internalized at time intervals of more than 1h, was not targeted to the same vesicular structures, but was sorted into distinct late compartments. We further found that fluorescent hydrazide dyes distributed not only to rapidly recycling endosomes but also to long-lived compartments that participated in plasma membrane repair after local laser injury. Our approach highlights the benefits of combining different fluid-phase markers in conjunction with membrane dyes in simultaneous and sequential application modus for investigating vesicle traffic, especially in organisms, which are still refractory to genetic transformation like characean algae. [ABSTRACT FROM AUTHOR]

Filamentous fungi grow by adding cell wall and membrane exclusively at the apex of tubular structures called hyphae. Growth was previously believed to occur only through exocytosis at the Spitzenkörper, an organised body of secretory macro- and microvesicles found only in growing hyphae. More recent work has indicated that an area deemed the sub-apical collar is enriched for endocytosis and is also required for hyphal growth. It is now generally believed that polarity of filamentous fungi is achieved through the balancing of the processes of endocytosis and exocytosis at these two areas. This review is an update on the current progress and understanding surrounding the occurrence of endocytosis and its spatial regulation as they pertain to growth and pathogenicity in filamentous fungi. [ABSTRACT FROM AUTHOR]

Infection by positive-strand RNA viruses induces extensive remodeling of the host endomembrane system in favor of viral replication and movement. The integral membrane protein 6K2 of potyviruses induces the formation of membranous virus replication vesicles at the endoplasmic reticulum exit site (ERES). The intracellular trafficking of 6K2-induced vesicles along with microfilaments requires the vesicular transport pathway, actomyosin motility system, and possibly post-Golgi compartments such as endosomes as well. Recent studies have shown that endocytosis is essential for the intracellular movement of potyviruses from the site of viral genome replication/assembly site to plasmodesmata (PD) to enter neighboring cells. In this chapter, we describe a detailed protocol of how to use endomembrane trafficking pathway-specific chemical inhibitors and organelle-selective fluorescence dye to study the trafficking of potyviral proteins and potyvirus-induced vesicles and to unravel the role of endocytosis and the endocytic pathway in potyvirus infection in Nicotiana benthamiana plants., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Autophagy is an essential system for degrading and recycling cellular components for survival during starvation conditions. Under sucrose starvation, application of a papain protease inhibitor E-64d to the Arabidopsis root and tobacco BY-2 cells induced the accumulation of vesicles, labeled with a fluorescent membrane marker FM4-64. The E-64d–induced vesicle accumulation was reduced in the mutant defective in autophagy-related genes ATG2 , ATG5 , and ATG7 , suggesting autophagy is involved in the formation of these vesicles. To clarify the formation of these vesicles in detail, we monitored time-dependent changes of tonoplast, and vesicle accumulation in sucrose-starved cells. We found that these vesicles were derived from the tonoplast and produced by microautophagic process. The tonoplast proteins were excluded from the vesicles, suggesting that the vesicles are generated from specific membrane domains. Concanamycin A treatment in GFP-ATG8a transgenic plants showed that not all FM4-64–labeled vesicles, which were derived from the tonoplast, contained the ATG8a-containing structure. These results suggest that ATG8a may not always be necessary for microautophagy. [ABSTRACT FROM AUTHOR]

Tip growth is a common strategy for the rapid elongation of cells to forage the environment and/or to target to long-distance destinations. In the model tip growth system of Arabidopsis pollen tubes, several small-molecule hormones regulate their elongation, but how these rapidly diffusing molecules control extremely localized growth remains mysterious. Here we show that the interconvertible salicylic acid (SA) and methylated SA (MeSA), well characterized for their roles in plant defense, oppositely regulate Arabidopsis pollen tip growth with SA being inhibitory and MeSA stimulatory. The effect of SA and MeSA was independent of known NPR3/NPR4 SA receptor-mediated signaling pathways. SA inhibited clathrin-mediated endocytosis in pollen tubes associated with an increased accumulation of less stretchable demethylated pectin in the apical wall, whereas MeSA did the opposite. Furthermore, SA and MeSA alter the apical activation of ROP1 GTPase, a key regulator of tip growth in pollen tubes, in an opposite manner. Interestingly, both MeSA methylesterase and SA methyltransferase, which catalyze the interconversion between SA and MeSA, are localized at the apical region of pollen tubes, indicating of the tip-localized production of SA and MeSA and consistent with their effects on the apical cellular activities. These findings suggest that local generation of a highly diffusible signal can regulate polarized cell growth, providing a novel mechanism of cell polarity control apart from the one involving protein and mRNA polarization. [ABSTRACT FROM AUTHOR]

The localization of proteins to the Golgi complex is a dynamic process requiring sorting signals in the cytosolic domains of resident Golgi proteins and retrograde vesicular trafficking. Disruptions in these signals or in the retrograde pathways often lead to mislocalization of Golgi proteins to the vacuole in budding yeast. The extent of vacuolar mislocalization can be quantified through colocalization of GFP-tagged Golgi proteins with fluorescent dyes that mark either the vacuole limiting membrane or the vacuole lumen. Manders' colocalization coefficient (MCC) is a useful tool for quantifying the degree of colocalization. However, the dilution of fluorescence signal intensity that occurs when GFP-tagged Golgi proteins mislocalize to the much larger vacuole is problematic for thresholding the images prior to calculating the MCC. In this chapter, we describe the use of Multi-Otsu thresholding in ImageJ to quantify the degree of GFP-tagged protein mislocalization to the vacuole. Furthermore, these methods can be applied to other colocalization events within the cell., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1's implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole. [ABSTRACT FROM AUTHOR]

Electroendocytosis (EED), i.e. electric field-induced endocytosis, is a technique for bio-molecule and drug delivery to cells using a pulsed electric field lower than that applied in electroporation (EP). Different from EP in which nanometer-sized electropores appear on the plasma membrane lipid bilayer, EED induces cell membrane internalization and fission via endocytotic vesicles. In this study, we conduct comprehensive experimental study on the EED of HeLa cells using a micro chip and the corresponding endocytotic vesicles were visualized and investigated by using FM4-64 fluorescent dye and in situ fluorescence microscopy. The uptake of molecules by the EED of cells was characterized by average intracellular fluorescent intensity from a large number (>2,000) of single cells. The EED efficiency was determined as a function of three electric parameters (electric field strength, pulse duration, total electric treatment time). The EED efficiency as a function of electric field strength clearly shows biphasic characteristics at different experimental conditions. The EED experiments using cytoskeleton inhibitors illustrate unique mechanisms distinct from EP. This study provides a foundation for further on-chip study of the time-dependent mechanism of EED at the single-cell level. [ABSTRACT FROM AUTHOR]

Eisosomes are large immobile assemblies at the cortex of a cell under the membrane compartment of Can1 (MCC) in yeast. Slm1 has recently been identified as an MCC component that acts downstream of Mss4 in a pathway that regulates actin cytoskeleton organization in response to stress. In this study, we showed that inactivation of Slm proteins disrupts proper localization of the primary eisosome marker Pil1, providing evidence that Slm proteins play a role in eisosome organization. Furthermore, we found that slm mutant cells exhibit actin defects in both the ability to polarize cortical F-actin and the formation of cytoplasmic actin cables even at the permissive temperature (30°C). We further demonstrated that the actin defect accounts for the slow traffic of FM4-64-labelled endosome in the cytoplasm, supporting the notion that intact actin is essential for endosome trafficking. However, our real-time microscopic analysis of Abp1-RFP revealed that the actin defect in slm cells was not accompanied by a noticeable defect in actin patch internalization during receptor-mediated endocytosis. In addition, we found that slm cells displayed impaired membrane recycling and that recycling occurred in an actin-independent manner. Our data provide evidence for the requirement of Slm proteins in eisosome organization and endosome trafficking and recycling. [ABSTRACT FROM AUTHOR]

This report describes the analyses of three Candida albicans genes that encode Src Homology 3 (SH3)-domain proteins. Homologs in Saccharomyces cerevisiae are encoded by the SLA1, NBP2, and CYK3 genes. Deletion of CYK3 in C. albicans was not feasible, suggesting it is essential. Promoter shutdown experiments of CaCYK3 revealed cytokinesis defects, which are in line with the localization of GFP-tagged Cyk3 at septal sites. Deletion of SLA1 resulted in strains with decreased ability to form hyphal filaments. The number of cortical actin patches was strongly reduced in Δsla1 strains during all growth stages. Sla1-GFP localizes in patches that are found concentrated at the hyphal tip. Deletion of the first two SH3-domains of Sla1 still resulted in cortical localization of the truncated protein. However, the actin cytoskeleton in this strain was aberrant like in the Δsla1 deletion mutant indicating a function of these SH3 domains to recruit actin nucleation to sites of endocytosis. Deletion of NBP2 resulted in a defect in vacuolar fusion in hyphae. Germ cells of Δnbp2 strains lacked a large vacuole but initiated several germ tubes. The mutant phenotypes of Δnbp2 and Δsla1 could be corrected by reintegration of the wild-type genes. [ABSTRACT FROM AUTHOR]

Abstract: Mammalian dynamin is responsible for scission of endocytic vesicles from the plasma membrane. A previous study showed that Vps1, a yeast dynamin-like protein, plays an important role in pheromone receptor internalization (; J. Cell Sci. 117, 3839–3853). However, the details of how Vps1 acts in various phases of endocytosis including early internalization of the endocytic vesicle are poorly understood. To investigate the potential roles of Vps1 in both endocytic vesicle formation/maturation on the plasma membrane and endocytic vesicle internalization, time-lapse fluorescent images of GFP-tagged endocytic markers in live cells were analyzed using a particle tracking software. The loss of Vps1 leads to a robust increase in the lifespan of newly forming cortical endocytic vesicles carrying Las17-GFP, Ede1-GFP, Sla1-GFP, and Abp1-GFP, indicating that Vps1 is required for the proper assembly and maturation of endocytic vesicles. Particle track analysis revealed that Abp1-GFP vesicles in vps1 null cells moved a relatively short distance away from the cell membrane due to their non-directional movement. Furthermore, we found that the GTPase and the GED domains of Vps1 are required for the proper endocytic function of Vps1. Our tracking analysis data also revealed that the post-internalized vesicle motility en route to the vacuole was decreased significantly, perhaps due to severe disruption of the actin cables in Vps1 mutant cells. [Copyright &y& Elsevier]

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all ∼110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis. [ABSTRACT FROM AUTHOR]

The phragmoplast is a special apparatus that functions in establishing a cell plate in dividing plant cells. It is known that microfilaments (MFs) are involved in constituting phragmoplast structure, but the dynamic distribution and role of phragmoplast MFs are far from being understood. In this study, the precise structure and dynamics of MFs during the initiation and the late lateral expansion of the phragmoplast were observed by using a tobacco BY-2 cell line stably expressing the microfilament reporter construct GFP-fABD2. Three-dimensional imaging showed that the phragmoplast MFs were initiated by two populations of MFs emerging between the reconstituting daughter nuclei at anaphase, which migrated to the mid-zone and gave rise to two layers of microfilament arrays. FM4-64 stained vesicles accumulated and fused with the cell plate between the two populations of MFs. The two layers of microfilament arrays of phragmoplast with ends overlapped always surrounded the centrifugally expanding cell plate. Partial disruption of MFs at metaphase by low concentration of latrunculin B resulted in the inhibition of the cell plate consolidation and the blockage of cell plate lateral expansion, whereas high concentration of latrunculin B restrained the progression of the cell cycle. Treating the cell after the initiation of phragmoplast led to the cease of the expansion of the cell plate. Our observations provide new insights into the precise structure and dynamics of phragmoplast MFs during cytokinesis and suggest that dynamic phragmoplast MFs are important in cell plate formation. [ABSTRACT FROM AUTHOR]

Abstract: We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by “click” labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions. [Copyright &y& Elsevier]

The amphiphilic dye FM4–64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4–64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4–64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4–64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4–64 interacts with. [ABSTRACT FROM AUTHOR]

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis. [ABSTRACT FROM PUBLISHER]

We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-( p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620–650 nm in contrast to 480–520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual-excitation imaging of FM4-64 in relation to lamina-associated polypeptide-1–green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long-wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4-64 as the first vital dye sensitive to the NE environment opens new ways for real-time visualization and functional studies of the NE. [ABSTRACT FROM AUTHOR]

Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (GFP)–fimbrin (BY-GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)–fimbrin in tobacco BY-2 cells stably expressing GFP–AtVam3p (BY-GV7) and BY-2 cells stably expressing γ-tonoplast intrinsic protein (γ-TIP)–GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating region. Three-dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for the acto-myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner. [Copyright &y& Elsevier]

In the fission yeast, Schizosaccharomyces pombe, uptake of the fluorescent styryl dye FM4-64 via the endocytic pathway to the vacuole was localised to the poles of growing, interphase cells and to the cell equator during cell division, regions of cell wall deposition that are rich in actin. When the pattern of growth or the plane of cytokinesis was altered, the relationship between the actin cytoskeleton and the site of endocytosis was maintained. Transfer of the label to the vacuolar membrane was dependent upon the Rab GTPase Ypt7 and, hence, vesicle fusion. Endocytic vesicles transiently colocalised with actin patches and endocytosis was inhibited in mutants that affected actin patch integrity and by the actin inhibitor latrunculin A. Concentrations of latrunculin that removed actin cables but left patches unaffected had no effect on endocytosis at the poles, but abolished endocytosis at the cell equator. Equatorial, but not polar, end ocytosis was also inhibited in cells lacking the formin For3 (which have selectively destabilised actin cables), in mutants of the exocyst complex and in cells treated with brefeldin A. Differential effects on endocytosis at the cell poles and equator were also observed in the actin mutant cps8 and the Arp2/3 complex mutant arp2. The redirection of endocytosis from the cell poles to the cell equator in M phase coincided with the anaphase separation of sister chromatids and was abolished in the septation initiation network (SIN) mutants cdc7, sid1 and sid2, demonstrating that the spatial reorganisation of the endocytic pathway in the S. pombe cell cycle requires a functional SIN pathway. We conclude that endocytosis in fission yeast has two distinct components, both of which are actin-based, but which are mechanistically distinct, as well as being spatially and temporally separated in the S. pombe cell cycle. [ABSTRACT FROM AUTHOR]

Boron (B) is essential for plants but toxic when present in excess. Arabidopsis thaliana BOR1 is a B exporter for xylem loading and is essential for efficient B translocation from roots to shoots under B limitation. B translocation to shoots was enhanced under B limitation in WT but not in bor1-1 mutant plants. The enhanced translocation was suppressed upon resupply of high levels of B within several hours. Unlike a number of transporters for essential mineral nutrients, BOR1 mRNA accumulation was not strongly affected by B conditions. However, accumulation of a constitutively expressed BOR1-GFP fusion protein was elevated under conditions of limited B supply. Upon resupply of high levels of B, BOR1-GFP was degraded within several hours. These findings demonstrate that posttranscriptional mechanisms play a major role in regulation of BOR1 accumulation. Confocal laser scanning microscopy of root tip cells showed that BOR1-GFP is localized to the plasma membrane under B limitation. Shortly after B application, the protein was observed in dot-like structures in the cytoplasm before degradation. Colocalization studies of the fusion protein with an endocytic tracer FM4-64 and an endosomal Rab-GTPase Ara7 fused to monomeric red fluorescent protein suggested that BOR1 is transferred from the plasma membrane via the endosomes to the vacuole for degradation. These results establish that endocytosis and degradation of BOR1 are regulated by B availability, to avoid accumulation of toxic levels of B in shoots under high-B supply, while protecting the shoot from B deficiency under B limitation. [ABSTRACT FROM AUTHOR]

Neurotrophins are a family of secreted neuronal survival and plasticity factors comprising NGF, BDNF, neurotrophin-3 (NT-3), and NT-4. Whereas synaptic secretion of BDNF has been described, the routes of intracellular targeting and secretion of NGF, NT-3, and NT-4 in neurons are poorly understood. To allow for a direct comparison of intracellular targeting and release properties, all four mammalian neurotrophins were expressed as green fluorescent protein fusion proteins in cultured rat hippocampal neurons. We show that BDNF and NT-3 are targeted more efficiently to dendritic secretory granules of the regulated pathway of secretion (BDNF, in 98% of cells; NT-3, 85%) than NGF (46%) and NT-4 (23%). For all NTs, the remaining cells showed targeting to the constitutive secretory pathway. Fusing the BDNF pre-pro sequence to NT-4 directed NT-4 more efficiently to the regulated pathway of secretion. All neurotrophins, once directed to the regulated secretion pathway, were detected near synapsin I-positive presynaptic terminals and colocalized with PSD-95-DsRed (postsynaptic density-95-Discosoma red), suggesting postsynaptic targeting of the neurotrophins to glutamatergic synapses. Depolarization-induced release of all neurotrophins from synaptic secretory granules was slow (delay in onset, 10-30 s; τ = 120-307 s) compared with transmitter release kinetics monitored with FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] destaining (onset, <5 s; τ = 13 ± 2 s). Among the neurotrophins, NT-4 secretion was most rapid but still proceeded 10 times more slowly than transmitter secretion. Preincubation of neurons with monensin (neutralizing intragranular pH, thus solubilizing the peptide core) increased the speed of secretion of BDNF, NGF, and NT-3 to the value of NT-4. These data suggest that peptide core dissolution in secretory granules is the critical determinant of the speed of synaptic secretion of all mammalian NTs and that the speed of release is not compatible with fast transmitter-like actions of neurotrophins. [ABSTRACT FROM AUTHOR]

Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkörper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkörper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkörper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkörper and polarisome are distinct structures, that the polarisome and Spitzenkörper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae. [ABSTRACT FROM AUTHOR]

In long-term time-lapse imaging of living cells, where recording takes several minutes or longer, a drift of focus may be significant. Focus-drift is due to the slippage in the microscope focus mechanism and/or the thermal gradients in the microscope. Software and hardware solutions may be introduced to correct for the focus-drift. Some autofocus techniques measure position of the specimen by sensing the light or sound reflected from a well-defined surface, such as the microscope slide. An autofocusing approach, where a focus measure is computed for images acquired at different objective positions is less appropriate in confocal microscopy, since more than one section is in focus. To correct for the focal-drift in long-term time-lapse confocal imaging, we acquired an image stack of the specimen periodically. The software calculated Pearson's correlation coefficient between each image in the z-stack and the reference image in the stack, which was selected at the beginning of the experiment. The maximal correlation coefficient of pixel intensities was taken to identify the image, which corresponded to the focal plane of the reference image. To test our approach, we used confocal images of living rat lactotroph cells, which discharged preloaded green fluorescent probe from a single secretory granule. Simultaneously, an extracellularly applied FM 4-64 red fluorescent probe loaded the discharging vesicle and the plasma membrane. We show that our approach is appropriate to correct for focal-drift in long term time-lapse imaging and analysis of living cells. [ABSTRACT FROM AUTHOR]

Autolysosomes accumulate in tobacco cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We characterized these plant autolysosomes using fluorescent dyes and green fluorescent protein (GFP). Observation using the endocytosis markers, FM4-64 and Lucifer Yellow CH, suggested that there is a membrane flow from the plasma membrane to autolysosomes. Using these dyes as well as GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP fusion proteins as markers of the central vacuole, we found transport of components of the central vacuole to autolysosomes. Thus endocytosis and the supply from the central vacuole may contribute to the formation of autolysosomes. [ABSTRACT FROM AUTHOR]

During exocytosis, the lumen of secretory vesicles connects with the extracellular space. In some vesicles, this connection closes again, causing the vesicle to be recaptured mostly intact. The degree to which the bilayers of such vesicles mix with the plasma membrane is unknown. Work supporting the kiss-and-run model of transient exocytosis implies that synaptic vesicles allow neither lipid nor protein to escape into the plasma membrane, suggesting that the two bilayers never merge. Here, we test whether neuroendocrine granules behave similarly. Using two-color evanescent field microscopy, we imaged the lipid probe FM4-64 and fluorescent proteins in single dense core granules. During exocytosis, granules lost FM4-64 into the plasma membrane in small fractions of a second. Although FM4-64 was lost, granules retained the membrane protein, GEP-phogrin. By using GFP-phogrin as a probe for resealing, it was found that even granules that reseal lose FM4-64. We conclude that the lipid bilayers of the granule and the plasma membrane become continuous even when exocytosis is transient. [ABSTRACT FROM AUTHOR]

The evidence and arguments for and against the occurrence of endocytosis in fungal hyphae are summarized. The balance of evidence is in favour of the existence of endocytosis. This is supported by an analysis of the recently sequenced Neurospora genome which strongly suggests that this fungus possesses the complex protein machinery required to conduct endocytosis. [Copyright &y& Elsevier]

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2–3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection. [Copyright &y& Elsevier]

Reports of the existence of endocytosis in filamentous fungi have been conflicting and inconclusive. For this reason, we have tested three independent markers in Neurospora crassa: the electron opaque marker lanthanum (La) and the fluorescent probes Lucifer yellow (LY) and FM4-64. Both La and LY were endocytosed by Saccharomyces cerevisiae cells, which were used as positive controls for endocytosis, but the probes did not accumulate in N. crassa hyphae. Only FM4-64 became internalized into N. crassa hyphae, but it induced abnormal changes in membrane systems and its internalization could be explained by mechanisms other than endocytosis. Together, our results suggest that endocytosis does not occur in N. crassa hyphae and question whether the styryl dyes do in fact reliably report normal endocytosis in filamentous fungi. [Copyright &y& Elsevier]

Abstract The effect of fibroblast growth factor-2 (FGF-2) on synapse formation was investigated using rat cultured hippocampal neurons. Treatment with FGF-2 (0.4–10 ng/mL) for 6 days enhanced synaptogenesis on these neurons by ∼50%, as determined by counting puncta immunostained for presynaptic- or postsynaptic-specific proteins. This enhancement was statistically significant, and was abolished by a specific inhibitor of mitogen-activated protein kinase (MAPK). The majority of neurons expressed FGF receptors (types 1–3) abundantly on the membrane of somata, dendrites, and growth cones, and in these regions phosphorylation of MAPK was enhanced after FGF-2 application. Furthermore, our experiments showed that the majority of synapses formed in cultures containing FGF-2 were positive both for presynaptic proteins and postsynaptic excitatory synapse-specific proteins, and that these synapses had a similar capacity to recycle the fluorescent styryl dye FM4-64 as those in the control culture. These results indicate that: (i) FGF-2 increases excitatory synapses on hippocampal neurons by activating MAPK activity through FGF receptors; and (ii) synapses formed in FGF-2-treated culture are capable of cycling vesicles. [ABSTRACT FROM AUTHOR]

In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G2 phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G1 phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase. [ABSTRACT FROM PUBLISHER]

[Display omitted] • Nuclear-envelope invaginations are putative sites of lipofected-DNA nuclear access. • 3D confocal imaging of lipofected-DNA effective localization. • The lipid formulation modulates DNA accumulation at nuclear-envelope invaginations. Nuclear breakdown was found to be the dominant route for DNA entry into the nucleus in actively dividing cells. The possibility that alternative routes contribute to DNA entry into the nucleus, however, cannot be ruled out. Here we address the process of lipofection by monitoring the localization of fluorescently-labelled DNA plasmids at the single-cell level by confocal imaging in living interphase cells. As test formulation we choose the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE) with plasmidic DNA pre-condensed by means of protamine. By exploiting the spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br) we monitor the position of the nuclear envelope (NE), while concomitantly imaging the whole nucleus (by Hoechst) and the DNA (by Cy3 fluorophore) in a multi-channel 3D confocal imaging experiment. Reported results show that DNA clusters are typically associated with the NE membrane in the form of tubular invaginations spanning the nuclear environment, but not completely trapped within the NE invaginations, i.e. the DNA may use these NE regions as entry-points towards the nucleus. These observations pave the way to investigating the molecular details of the postulated processes for a better exploitation of gene-delivery vectors, particularly for applications in non-dividing cells. [ABSTRACT FROM AUTHOR]

Ca 2+ regulates a variety of cellular processes that are essential to maintain cell integrity and function. Different methods have been used to study these processes by increasing intracellular Ca 2+ levels. Here, we describe a protocol to initiate Ca 2+ -dependent membrane-related events, using laser ablation by near-infrared irradiation. This creates a rupture in the plasma membrane that allows the extracellular Ca 2+ to enter the cell and thereby induce a receptor-independent Ca 2+ increase. We report laser ablation protocols to study two different Ca 2+ -induced processes in human endothelial cells-membrane resealing and exocytosis of secretory granules called Weibel-Palade bodies (WPBs). Thus, laser ablation represents a technique that permits the analysis of different Ca 2+ -regulated processes at high spatiotemporal resolution in a controlled manner.

Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches., (© 2020 Wiley Periodicals LLC.)

Filamentous fungi grow by adding cell wall and membrane exclusively at the apex of tubular structures called hyphae. Growth was previously believed to occur only through exocytosis at the Spitzenkörper, an organised body of secretory macro- and microvesicles found only in growing hyphae. More recent work has indicated that an area deemed the sub-apical collar is enriched for endocytosis and is also required for hyphal growth. It is now generally believed that polarity of filamentous fungi is achieved through the balancing of the processes of endocytosis and exocytosis at these two areas. This review is an update on the current progress and understanding surrounding the occurrence of endocytosis and its spatial regulation as they pertain to growth and pathogenicity in filamentous fungi., Competing Interests: No potential conflict of interest was reported by the authors., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Endocytosis is an intracellular trafficking pathway that occurs in nutrient uptake, signal transduction and reconstruction of cell polarity and is conserved in eukaryotic cells. In fungi, endocytosis plays crucial roles in the physiology of hyphal growth and pathogenicity. vidence for endocytosis in filamentous fungi is detected by the membrane-selective dyes FM4-64. Cells of a range of filamentous fungal species readily take up these dyes. However, the method for endocytosis detection has not been well established in Magnaporthe oryzae . Here, we provide a protocol for tracking endocytosis in Magnaporthe oryzae ., Competing Interests: Competing interestsNo conflicts of interest or competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

In plants, the partitioning of daughter cells during cytokinesis is achieved via physical insertion of a membranous cell plate within the dividing parent cell. It is a cellular process of extensive protein secretion and membrane trafficking toward the plane of cell division and the cytoskeleton is an important facilitator of this process. A specialized cytoskeletal array termed phragmoplast expands centrifugally throughout cytokinesis and directs, mostly Golgi-derived vesicles that ultimately fuse to form the developing cell plate. The function of the phragmoplast in guiding cell plate synthesis has strongly motivated many scientists to monitor its dynamic behavior. In this chapter, we present an overview of basic principles and methods concerning the live imaging of cytokinetic plant cells using confocal laser scanning microscopy (CLSM) and the analysis of phragmoplast expansion.