The microtubule analog protein, FtsZ, in the endosymbiont of trypanosomatid protozoa

Blastocrithidia culicis and Crithidia deanei are trypanosomatids that harbor an endosymbiotic bacterium in their cytoplasm. In prokaryotes, numerous proteins are essential for cell division, such as FtsZ, which is encoded by filament-forming temperature-sensitive (fts) genes. FtsZ is the prokaryotic homolog of eukaryotic tubulin and is present in bacteria and archaea, and has also been identified in mitochondria and chloroplasts. FtsZ plays a key role in the initiation of cytokinesis. It self-assembles into the Z ring, which establishes the division plane during septation. In this study, immunoblotting analysis using a FtsZ polyclonal antibody, revealed a 40-kDa band characteristic of FtsZ in endosymbiont fractions and in whole trypanosomatid homogenates, but not in whole cell extracts of aposymbiotic strains. Confocal microscopy and ultrastructural analysis revealed a specific and dispersed labeling over the endosymbiont. Bars and ring-like structures, which are suggestive of the presence of Z-rings, were never observed, even during the division of the symbiont. This peculiar distribution of FtsZ may represent an arrangement of cytoskeleton protein intermediate between prokaryotic and eukaryotic cells. The endosymbiont ftsz gene was completely sequenced after amplification of DNA from symbiont-bearing trypanosomatids or from pure endosymbiont fractions, using PCR and specific primers. The sequences obtained from the endosymbionts from C. deanei and B. culicis were very similar, and were most closely related to bacteria from the genus Pseudomonas.     

An Improved Immunolabeling Method for Microtubular Cytoskeleton in Poplar (Populus nigra L) Free Nuclear Endosperm

We investigated immunocytochemical staining of microtubular cytoskeleton of free nuclear endosperm, a tissue which is particularly difficult to fix. This tissue requires fixation for 45 hr to preserve the integrity of the microtubular network after paraformaldehyde based fixation. Low glutaraldehyde concentration in the fixative and the ethanol dehydration retains β-tubulin antigenicity and the former improves preservation of tissue structure. An ethanol-free embedding method is recommended for immunocytochemical studies of ethanol sensitive target proteins.     

Muscle and nerve net organization in stalked jellyfish (Medusozoa: Staurozoa)

Staurozoan cnidarians display an unusual combination of polyp and medusa characteristics and their morphology may be informative about the evolutionary origin of medusae. We studied neuromuscular morphology of two staurozoans, Haliclystus ‘sanjuanensis ’ and Manania handi , using whole mount immunohistochemistry with antibodies against FMRFamide and α‐tubulin to label neurons and phalloidin to label muscles. All muscles appeared to lack striations. Longitudinal interradial muscles are probable homologues of stalk muscles in scyphopolyps, but in adult staurozoans they are elaborated to inwardly flex marginal lobes of the calyx during prey capture; these muscles are pennate in M. handi . Manubrial perradial muscles, like the manubrium itself, are an innovation shared with pelagic medusae and manubrial interradial muscles are shared with scyphozoan ephyra. Marginal muscles of M. handi displayed occasional synchronous contraction reminiscent of a medusa swim pulse, but contractions were not repetitive. The nerve net in both species showed regional variation in density and orientation of neurons. Some areas labeled predominantly by α‐tubulin antibodies (exumbrellar epidermis), other areas labeled exclusively by FMRFamide antibodies (dense plexus of neurites surrounding the base of secondary tentacles, neuronal concentration at the base of transformed primary tentacles; gastrodermal nerve net), but most areas showed a mix of neurons labeled by these two antibodies and frequent co‐labeling of neurons. Transformed primary tentacles had a concentration of FMRFamide‐immunoreactive neurons at their base that was associated with a pigment spot in M. handi; this is consistent with their homology with rhopalia of medusae, which are also derived from primary tentacles. The muscular system of these staurozoans embodies characteristics of both scyphopolyps and pelagic medusae. However, their nerve net is more polyp‐like, although marginal concentrations of the net associated with primary and secondary tentacles may facilitate the richer behavioral repertoire of staurozoans relative to polyps of other medusozoans. J. Morphol. 278:29–49, 2017. ©© 2016 Wiley Periodicals,Inc.     

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria¹, fungi² and parasites³. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent⁴. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase⁵. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility⁶.We describe methods to isolate Neutrophil Granulocytes from peripheral human blood⁷ and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.     

藻胆蛋白荧光探针及其标记

藻胆蛋白是一系列新型的荧光标记探针,具有优良的荧光特性,以藻胆蛋白荧光探针标记抗体还可用于血清可溶性抗原（或抗体）的荧光免疫检测,其标记方法可分为直接法和间接法。结合藻胆蛋白的特点,研究藻胆蛋白的标记方法有助于提高荧光免疫检测的灵敏度。     

Labeling of BSA and imaging of mouse T‐lymphocyte as well as mouse spleen tissue by l‐glutathione capped CdTe quantum dots

l ‐glutathione capped highly fluorescent CdTe quantum dots (QDs) were prepared by an aqueous approach and used as fluorescent labels to link albumin bovine serum (BSA) and rat anti‐mouse CD4, which was expressed on mouse T‐lymphocyte and mouse spleen tissue. The sharp and narrow emission peaks showed that the as‐prepared QDs have desirable dispersibility, uniformity and good fluorescence properties. Both CdTe–BSA and CdTe–CD4 conjugates showed an enhancement of fluorescence intensity over that of bare CdTe QDs. The experimental result of gel electrophoresis confirmed the successful conjugation of CdTe–BSA and CdTe–CD4. The fluorescent microscopic images of CdTe–CD4 labeled mouse T‐lymphocyte cells and mouse spleen tissue were compared with that obtained from fluorescein isothiocyanate labeling. It was demonstrated that the CdTe QDs‐based probe exhibited much better photostability and fluorescence intensity than fluorescein isothiocyanate, showing a good application potential in the immuno‐labeling of cells and tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Ordering of compartments in the yeast endocytic pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t-SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t-SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.     

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

Immunogold labeling of permeabilized whole‐mount cells or thin‐sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3‐dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation—but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze‐substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre‐embedding NANOGOLD‐silver immunocytochemistry. So obtained thin and semi‐thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.      

ANALYSIS OF MUCILAGE SECRETION AND EXCRETION IN MICRASTERIAS (CHLOROPHYTA) BY MEANS OF IMMUNOELECTRON MICROSCOPY AND DIGITAL TIME LAPSE VIDEO MICROSCOPY1

Two different, independent, and alternative modes of mucilage excretion were found in the unicellular green alga Micrasterias denticulata Bréb. under constant culture conditions. The cells were capable of either excreting mucilage over all their cell surface or they extruded mucilage from one of their polar ends, which enabled directed movement such as photoorientation or escape from unfavorable environmental conditions. By means of a polyclonal antibody raised against Micrasterias mucilage, the secretory pathway of Golgi derived mucilage vesicles from their origin to their discharge was analyzed by means of conventional and energy filtering TEM. Depending on the stage of the cell cycle, mucilage vesicles were subjected to maturation processes. This may occur either after they have been pinched off from the dictyosomes (e.g. during cell growth) or when still connected to trans‐Golgi cisternae, as in the case of interphase cells. Only fully grown mature vesicles contained mucilage in its final composition as indicated by antibody labeling. After fusion of mucilage vesicles with vacuoles, no immunolabeling was found in vacuoles, indicating that the vesicle content was digested. Mucilage vesicles fused with the plasma membrane in areas of cell wall pores but were also able to excrete mucilage at any site directly through the respective cell wall layer. This result disproves earlier assumptions that the pore apparatus in desmids are the only mucilage excreting areas at the cell surface. Both mechanisms, excretion through the pores and through the cell wall, lead to formation of mucilage envelopes covering the entire cell surface.     

Marked disparity between age-related changes in dopamine and other presynaptic dopaminergic markers in human striatum

Because age-related changes in brain dopaminergic innervation are assumed to influence human disorders involving dopamine (DA), we measured the levels of several presynpatic DAergic markers [DA, homovanillic acid, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT)] in post-mortem human striatum (caudate and putamen) from 56 neurologically normal subjects aged 1 day to 103 years. Striatal DA levels exhibited pronounced (2- to 3-fold) post-natal increases through adolescence and then decreases during aging. Similarly, TH and AADC increased almost 100% during the first 2 post-natal years; however, the levels of TH and, to a lesser extent, AADC then declined to adult levels by approximately 30 years of age. Although VMAT2 and DAT levels closely paralleled those of TH, resulting in relatively constant TH to transporter ratios during development and aging, a modest but significant decline (13%) in DAT levels was observed in only caudate during aging. This biphasic post-natal pattern of the presynaptic markers suggests that striatal DAergic innervation/neuropil appears to continue to develop well past birth but appears to become overelaborated and undergo regressive remodeling during adolescence. However, during adulthood, a striking discrepancy was observed between the loss of DA and the relative preservation of proteins involved in its biosynthesis and compartmentation. This suggests that declines in DA-related function during adulthood and senescence may be explained by losses in DA per se as opposed to DAergic neuropil.