Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image‐based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment‐targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large‐scale experimental designs with standard instruments.     

A Unique C‐terminal Domain Allows Retention of Matrix Metalloproteinase‐27 in the Endoplasmic Reticulum

Matrix metalloproteinase‐27 (MMP‐27) is poorly characterized. Sequence comparison suggests that a C‐terminal extension (CTE) includes a potential transmembrane domain as in some membrane‐type (MT)‐MMPs. Having noticed that MMP‐27 was barely secreted, we investigated its subcellular localization and addressed CTE contribution for MMP‐27 retention. Intracellular MMP‐27 was sensitive to endoglycosidase H. Subcellular fractionation and confocal microscopy evidenced retention of endogenous MMP‐27 or recombinant rMMP‐27 in the endoplasmic reticulum (ER) with locked exit across the intermediate compartment (ERGIC). Conversely, truncated rMMP‐27 without CTE accessed downstream secretory compartments (ERGIC and Golgi) and was constitutively secreted. CTE addition to rMMP‐10 (a secreted MMP) caused ER retention and blocked secretion. Addition of a PKA target sequence to the cytosolic C‐terminus of transmembrane MT1‐MMP/MMP‐14 led to effective phosphorylation upon forskolin stimulation, but not for MMP‐27, excluding transmembrane anchorage. Moreover, MMP‐27 was protected from digestion by proteinase K. Finally, MT1‐MMP/MMP‐14 but neither endogenous nor recombinant MMP‐27 partitioned in the detergent phase after Triton X‐114 extraction, indicating that MMP‐27 is not an integral membrane protein. In conclusion, MMP‐27 is efficiently retained within the ER due to its unique CTE, which does not lead to stable membrane insertion. This could represent a novel ER retention system.      

Visualizing Endocytotic Pathways at Transmission Electron Microscopy via Diaminobenzidine Photo-Oxidation by a Fluorescent Cell-Membrane Dye

The endocytotic pathway involves a complex, dynamic and interacting system of intracellular compartments. PKH26 is a fluorescent dye specific for long-lasting cell membrane labelling which has been successfully used for investigating cell internalization processes, at either flow cytometry or fluorescence microscopy. In the present work, diaminobenzidine photo-oxidation was tested as a procedure to detect PKH26 dye at transmission electron microscopy. Our results demonstrated that DAB photo-oxidation is a suitable technique to specifically visualise this fluorescent dye at the ultrastructural level: the distribution of the granular dark reaction product perfectly matches the pattern of the fluorescence staining, and the electron density of the fine precipitates makes the signal evident and precisely detectable on the different subcellular compartments involved in the plasma membrane internalization routes.Key words: Endocytosis, PKH26 dye, diaminobenzidine photo-oxidation, transmission electron microscopy     

Surface Trafficking of APP and BACE in Live Cells

Amyloid‐β (Aβ)‐peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aβ precursor protein (APP) by β‐site APP‐cleaving enzyme (BACE). Using live‐cell imaging of APP and BACE labeled with pH‐sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α‐secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual‐color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.      

The Arabidopsis Rab GTPase RabA4b localizes to the tips of growing root hair cells

Spatial and temporal control of cell wall deposition plays a unique and critical role during growth and development in plants. To characterize membrane trafficking pathways involved in these processes, we have examined the function of a plant Rab GTPase, RabA4b, during polarized expansion in developing root hair cells. Whereas a small fraction of RabA4b cofractionated with Golgi membrane marker proteins, the majority of this protein labeled a unique membrane compartment that did not cofractionate with the previously characterized trans-Golgi network syntaxin proteins SYP41 and SYP51. An enhanced yellow fluorescent protein (EYFP)-RabA4b fusion protein specifically localizes to the tips of growing root hair cells in Arabidopsis thaliana. Tip-localized EYFP-RabA4b disappears in mature root hair cells that have stopped expanding, and polar localization of the EYFP-RabA4b is disrupted by latrunculin B treatment. Loss of tip localization of EYFP-RabA4b was correlated with inhibition of expansion; upon washout of the inhibitor, root hair expansion recovered only after tip localization of the EYFP-RabA4b compartments was reestablished. Furthermore, in mutants with defective root hair morphology, EYFP-RabA4b was improperly localized or was absent from the tips of root hair cells. We propose that RabA4b regulates membrane trafficking through a compartment involved in the polarized secretion of cell wall components in plant cells.     

Effect of pH and ATP on the equilibrium density of lysosomes

Lysosomes are membrane bound structures that accumulate and hydrolyze material internalized by the endocytic pathway. A very conspicuous property of this subcellular compartment is its relatively high equilibrium density. The actual mechanism that regulates lysosomal density is poorly understood. In an attempt to gain knowledge on the factors that regulate lysosomal density we have assessed the equilibrium density of lysosomal markers after in vitro incubation of a lysosome-enriched subcellular fraction. Incubation at pH 6 for 10 min at 37°C causes a density shift of several lysosomal markers to light density regions of Percoll gradients. Addition of ATP was able to prevent the acid-induced density shift. Pretreatment of the vesicles with N-ethylmaleimide (NEM) or trypsin inhibited the effect of ATP. Working in intact cells, ATP depletion, a condition that causes cytoplasmic acidification, also decreases lysosomal density. The results indicate that at low pH lysosomal density is preserved by an active process that requires ATP and membrane associated proteins. © 1993 Wiley-Liss, Inc.     

Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal‐Like Compartments and the Golgi Complex

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post‐Golgi and it has been speculated that they pass through endosomal‐like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well‐established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant‐like secretory pathway .     

Application of Proteomic Marker Ensembles to Subcellular Organelle Identification

Compartmentalization of biological processes and the associated cellular components is crucial for cell function. Typically, the location of a component is revealed through a co-localization and/or co-purification with an organelle marker. Therefore, the identification of reliable markers is critical for a thorough understanding of cellular function and dysfunction. We fractionated macrophage-like RAW264.7 cells, both in the resting and endotoxin-activated states, into six fractions representing the major organelles/compartments: nuclei, mitochondria, cytoplasm, endoplasmic reticulum, and plasma membrane as well as an additional dense microsomal fraction. The identity of the first five of these fractions was confirmed via the distribution of conventional enzymatic markers. Through a quantitative liquid chromatography/mass spectrometry-based proteomics analysis of the fractions, we identified 50-member ensembles of marker proteins (“marker ensembles”) specific for each of the corresponding organelles/compartments. Our analysis attributed 206 of the 250 marker proteins (∼82%) to organelles that are consistent with the location annotations in the public domain (obtained using DAVID 2008, EntrezGene, Swiss-Prot, and references therein). Moreover, we were able to correct locations for a subset of the remaining proteins, thus proving the superior power of analysis using multiple organelles as compared with an analysis using one specific organelle. The marker ensembles were used to calculate the organelle composition of the six above mentioned subcellular fractions. Knowledge of the precise composition of these fractions can be used to calculate the levels of metabolites in the pure organelles. As a proof of principle, we applied these calculations to known mitochondria-specific lipids (cardiolipins and ubiquinones) and demonstrated their exclusive mitochondrial location. We speculate that the organelle-specific protein ensembles may be used to systematically redefine originally morphologically defined organelles as biochemical entities.One of the basic concepts of cell biology is compartmentalization of the cellular processes within subcellular structures, termed organelles. Organelles were originally identified in the 19th century as the morphological entities that are still reflected in their names (e.g. “nucleus” from the Latin “little nut,” “mitochondria” from the Greek “thread” + “grain,” or “reticulum” from the Latin “little net”). Later, the progress of biochemistry made it possible to assign to the various organelles their specific biological functions. Thus, detailed information about the location of biochemical reactions became crucial for the understanding of their roles in cell function or dysfunction. Current technology allows the location of a cell component (a protein or a metabolite) to be linked directly to a morphologically defined organelle (or even a suborganellar compartment) by using electron microscopy. However, more typically, the location of a component is determined on the basis of its co-localization with a known marker for the organelle or subcellular compartment. This co-localization can be either visualized microscopically (imaging approach) to preserve some degree of morphological information or determined through co-purification of the component and the marker in a subcellular fractionation (biochemical approach).For both the imaging and the biochemical approaches, optimal organelle markers are of the utmost importance. Conventional markers include proteins, DNA (for nucleus), and even physical/chemical parameters (electric potential for mitochondria and acidic pH for lysosomes). Protein markers are assayed using either an interaction with specific antibodies or their enzymatic activities. Unfortunately, the former is typically non-quantitative, whereas the latter, although semiquantitative, is subject to interference from multiple parameters of the environment as well as substrate and product sharing with non-marker proteins. For a biochemical approach, tightness of the anchoring of a marker to the corresponding organelle is also an issue. Moreover, an inherent problem is that most proteins are located in several organelles/compartments, which may result in false localization conclusions.Our goal was to identify specific, reliable, and universal protein markers for major subcellular organelles/compartments. The following principles were chosen as the basis for our approach. First, the search had to be conducted without a preconceived notion of the nature of the markers (e.g. we did not expect to necessarily confirm conventional markers as optimal). Second, the search had to be conducted in all major organelles/compartments simultaneously. Third, the aim was to identify relatively large panels (ensembles) of markers as opposed to the best single marker. The last two principles allowed us to address the problem of multiple locations of potential marker proteins. Some of them can be eliminated as markers; for others, the impact of multiple locations on further analysis can be negated by averaging of the data for large numbers of proteins (derivation of marker ensembles).To meet these goals, we performed a complete “quantitative” proteomics analysis of all major subcellular fractions in a single cell type. Numerous reports have focused on the proteomes of specific organelles or interrelated sets of organelles in various cell types (for reviews, see Refs. 1 and 2). However, a need for an integral systematic study in a single cell type has been evident for some time (2), and the present study is the first step aimed at addressing this need.The marker ensembles that we identified from the proteome data were used to quantify the composition of the subcellular fractions. It is becoming appreciated that a physical association of various organelles makes it next to impossible to completely separate the organelles and obtain pure fractions acceptable for detailed proteomics analysis (e.g. see Ref. 3). Therefore, correlative approaches such as protein correlation profiling (1, 3, 4) and localization of organelle proteins by isotope tagging (5, 6) have been suggested to address this problem. These approaches allowed the assignment of protein locations based on co-localization with known markers in a density gradient (1, 4–6) or in multiple fractions (7). We took this approach a step further and derived a quantitative composition of the fractions based on the distribution of the marker ensembles. Furthermore, this enabled us to calculate levels of various components (lipids and proteins) in pure organelles from experimental data obtained with less than pure fractions.The choice of a particular cell type for this study was somewhat arbitrary, and the resulting marker ensembles were optimal for the cell type for which they were generated; of course, they may have to be adjusted to be adapted for other cell types. We chose macrophage cells partly because this study was an integral part of a larger subcellular lipidomics/proteomics study under the auspices of the Lipid Metabolites and Pathways Strategy (LIPID MAPS Consortium). The macrophage plays a central role in inflammation and innate and adaptive immunity. The macrophage detects and attacks pathogens and orchestrates a host response by sending signals to other cells and tissues; in this process, the macrophage itself transits from a resting to an activated state. These two states differ vastly in function, morphology, and underlying protein expression profiles, and therefore, we aimed to identify marker ensembles that would be invariant with regard to the activation process.In the present study, the activation paradigm was treatment with Kdo₂1-lipid A. This defined, nearly homogeneous reagent is a form of lipopolysaccharide endotoxin that has all the essential biological properties of lipopolysaccharide (8).     

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'-diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake.     

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Background

RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.

Methodology/Principal Findings

Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.

Conclusions/Significance

A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.     

Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation

Background

Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited.

Methodology/Principal Findings

We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins.

Conclusion/Significance

These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.     

Phagocytosis of antibody‐opsonized tumor cells leads to the formation of a discrete vacuolar compartment in macrophages

Despite the rapidly expanding use of antibody‐based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody‐opsonized tumor cells is limited. Here we report the formation of a phagosome‐associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2‐positive cancer cells in the presence of the HER2‐specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome‐associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the destruction of phagocytosed, antibody‐opsonized cancer cells by macrophages.      

Microbe‐inducible trafficking pathways that control Toll‐like receptor signaling

The receptors of the mammalian innate immune system are designed for rapid microbial detection, and are located in organelles that are conducive to serve these needs. However, emerging evidence indicates that the sites of microbial detection are not the sites of innate immune signal transduction. Rather, microbial detection triggers the movement of receptors to regions of the cell where factors called sorting adaptors detect active receptors and promote downstream inflammatory responses. These findings highlight the critical role that membrane trafficking pathways play in the initiation of innate immunity to infection. In this review, we describe pathways that promote the microbe‐inducible endocytosis of Toll‐like receptors (TLRs), and the microbe‐inducible movement of TLRs between intracellular compartments. We highlight a new class of proteins called Transporters Associated with the eXecution of Inflammation (TAXI), which have the unique ability to transport TLRs and their microbial ligands to signaling‐competent regions of the cell, and we discuss the means by which the subcellular sites of signal transduction are defined.      

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans- Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans- TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL- containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.     

Purification and characterization of lysosomes from Chinese hamster ovary cells

Lysosomes were isolated from Chinese hamster ovary cells by fractionation of a postnuclear supernatant in consecutive density gradients. By marker enzyme analysis, the preparation was 63-fold enriched for lysosomes compared to the homogenate and contained at most trace amounts of marker activities for plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, cytosol, and mitochondria. The lysosomes were intact as indicated by greater than 95% latency of beta-hexosaminidase activity, and the yield was about 12% relative to the homogenate. By electron microscopy, the lysosomal preparation contained very few mitochondrial profiles. By cytochemistry, greater than 80% of the organelle profiles were positive for the native lysosomal marker, acid phosphatase, and profiles were positive for long-term internalized horseradish peroxidase, an endocytic marker for lysosomes. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lysosomal preparation displayed a unique pattern of polypeptides and was devoid of mitochondrial contamination. Lysosomes were fractionated into membrane and lumenal compartments by Na2CO3 treatment. Each compartment contained 20-30 distinct electrophoretic species ranging from 18 to 200 kDa. Each polypeptide could be assigned to either the membrane or lumenal compartment. A comparison of silver-stained polypeptides with those metabolically labeled with [35S]methionine indicated that, with the possible exception of an 18-kDa species, all of the major lysosomal polypeptides in both compartments were derived by endogenous synthesis in these exponentially growing fibroblasts.     

A multi‐colour/multi‐affinity marker set to visualize phosphoinositide dynamics in Arabidopsis

Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of total phospholipids, but are involved in many regulatory processes, such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, providing a unique composition for these compartments and contributing to their identity. The precise subcellular localization and dynamics of most PIP species is not fully understood in plants. Here, we designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana. Analysis of this multi‐affinity ‘PIPline’ marker set revealed previously unrecognized localization of various PIPs in root epidermis. Notably, we found that PI(4,5)P₂ is able to localize PIP₂‐interacting protein domains to the plasma membrane in non‐stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post‐Golgi/endosomal compartments, and the lowest concentration in the Golgi. Finally, we also found a similar gradient of PI3P from high in late endosomes to low in the tonoplast. Our library extends the range of available PIP biosensors, and will allow rapid progress in our understanding of PIP dynamics in plants.     

Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging

In fluctuation‐based optical nanoscopy, investigating high‐density labeled subcellular structures with high fidelity has been a significant challenge. In this study, based on super‐resolution radial fluctuation (SRRF) microscopy, the joint tagging (JT) strategy is employed to enable fast high‐density nanoscopic imaging and tracking. In fixed cell experiment, multiple types of quantum dots with distinguishable fluorescence spectra are jointly tagged to subcellular microtubules. In each spectral channel, the decrease in labeling density guarantees the high‐fidelity super‐resolution reconstruction using SRRF microscopy. Subsequently, the combination of all spectral channels achieves high‐density super‐resolution imaging of subcellular microtubules with a resolution of ~62 nm using JT assisted SRRF technique. In the live‐cell experiment, 3‐channel JT is utilized to track the dynamic motions of high‐density toxin‐induced lipid clusters for 1 minute, achieving the simultaneous tracking of many individual toxin‐induced lipid clusters spatially distributed significantly below the optical diffraction limit in living cells.      

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum