scholarly journals Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance

Science ◽  
2019 ◽  
Vol 367 (6475) ◽  
pp. 301-305 ◽  
Author(s):  
Spencer A. Freeman ◽  
Stefan Uderhardt ◽  
Amra Saric ◽  
Richard F. Collins ◽  
Catherine M. Buckley ◽  
...  
Keyword(s):  
Immune Surveillance ◽  
Extracellular Fluid ◽  
Volume Ratio ◽  
Underlying Mechanism ◽  
Endocytic Pathway ◽  
Curvature Sensing ◽  
Monovalent Ion ◽  
Endocytic Traffic

Despite ongoing (macro)pinocytosis of extracellular fluid, the volume of the endocytic pathway remains unchanged. To investigate the underlying mechanism, we used high-resolution video imaging to analyze the fate of macropinosomes formed by macrophages in vitro and in situ. Na+, the primary cationic osmolyte internalized, exited endocytic vacuoles via two-pore channels, accompanied by parallel efflux of Cl− and osmotically coupled water. The resulting shrinkage caused crenation of the membrane, which fostered recruitment of curvature-sensing proteins. These proteins stabilized tubules and promoted their elongation, driving vacuolar remodeling, receptor recycling, and resolution of the organelles. Failure to resolve internalized fluid impairs the tissue surveillance activity of resident macrophages. Thus, osmotically driven increases in the surface-to-volume ratio of endomembranes promote traffic between compartments and help to ensure tissue homeostasis.

scholarly journals Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion

2016 ◽  
Vol 214 (5) ◽  
pp. 517-527 ◽  
Author(s):  
Lee Dolat ◽  
Elias T. Spiliotis
Keyword(s):  
Cell Metabolism ◽  
Extracellular Fluid ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Septin Gtpases ◽  
And Function ◽  
Large Clusters ◽  
Blocking Antibodies

Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate–dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes.

Nanoporous Gold: A Biomaterial for Microfabricated Drug-Delivery Platforms

2012 ◽  
Vol 1415 ◽  
Author(s):  
Erkin Seker ◽  
Yevgeny Berdichevsky ◽  
Kevin J. Staley ◽  
Martin L. Yarmush
Keyword(s):  
Drug Delivery ◽  
Electrochemical Sensors ◽  
Volume Ratio ◽  
Nanostructured Material ◽  
Nanoporous Gold ◽  
Biological Processes ◽  
Au Thin Films

ABSTRACTNanoporous gold (np-Au) is a promising nanostructured material with many desirable properties, including large surface area-to-volume ratio, corrosion resistance, high conductivity, and well-studied thiol-based surface chemistry. While np-Au has been used in a variety of applications, from fuel cells to electrochemical sensors, its interface with biology, where many of its exciting applications lie, is surprisingly non-existent. This paper reports on drug delivery from np-Au thin films for modifying cell proliferation in situ. We expect that establishing np-Au as a biomaterial with drug delivery capabilities will create new opportunities for engineering advanced BioMEMS devices that can monitor and modulate biological processes in both in vitro and in vivo settings.

Nav1.5 sodium channels in macrophages in multiple sclerosis lesions

2012 ◽  
Vol 19 (5) ◽  
pp. 532-542 ◽  
Author(s):  
Joel A Black ◽  
Jia Newcombe ◽  
Stephen G Waxman
Keyword(s):  
Multiple Sclerosis ◽  
Protein A ◽  
Proteolipid Protein ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Myelin Degradation ◽  
Active Multiple Sclerosis

Background: Macrophages are dynamic participants in destruction of white matter in active multiple sclerosis (MS) plaques. Regulation of phagocytosis and myelin degradation along endosomal pathways in macrophages is highly-orchestrated and critically-dependent upon acidification of endosomal lumena. Evidence from in vitro studies with macrophages and THP-1 cells suggests that sodium channel Nav1.5 is present in the limiting membrane of maturing endosomes where it plays a prominent role in the accumulation of protons. However, a contribution of the Nav1.5 channel to macrophage-mediated events in vivo has not been demonstrated. Method: We examined macrophages within active MS lesions by immunohistochemistry to determine whether Nav1.5 is expressed in these cells in situ and, if expressed, whether it is localized to specific compartments along the endocytic pathway. Results: Our results demonstrate that Nav1.5 is expressed within macrophages in active MS lesions, and that it is preferentially expressed in late endosomes and phagolysosomes (Rab7+, LAMP-1+), and sparsely expressed in early (EEA-1+) endosomes. Triple-immunolabeling studies showed localization of Nav1.5 within Rab7+ endosomes containing proteolipid protein, a myelin marker, in macrophages within active MS plaques. Conclusions: These observations support the suggestion that Nav1.5 contributes to the phagocytic pathway of myelin degradation in macrophages in vivo within MS lesions.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

CONSERVATION OF RARE SPECIES PLANTS AND LICHENS IN SITU WITH PLANNED ANTHROPOGENIC ACTIVITIES: BASIC APPROACHES AND IMPLEMENTATION PRINCIPLES

2018 ◽  
Vol 12 (7-8) ◽  
pp. 38-45
Author(s):  
A. N. EFREMOV ◽  
N. V. PLIKINA ◽  
T. ABELI
Keyword(s):  
Rare Species ◽  
Technology Implementation ◽  
Technology Development ◽  
Anthropogenic Activities ◽  
Natural Habitat ◽  
Local Population ◽  
Main Stage ◽  
Social Risks

Rare species are most vulnerable to man-made impacts, due to their biological characteristics or natural resource management. As a rule, the economic impact is associated with the destruction and damage of individual organisms, the destruction or alienation of habitats. Unfortunately, the conservation of habitat integrity is an important protection strategy, which is not always achievable in the implementation of industrial and infrastructural projects. The aim of the publication is to summarize the experience in the field of protection of rare species in the natural habitat (in situ), to evaluate and analyze the possibility of using existing methods in design and survey activities. In this regard, the main methodological approaches to the protection of rare species in the natural habitat (in situ) during the proposed economic activity were reflected. The algorithm suggested by the authors for implementing the in situ project should include a preparatory stage (initial data collection, preliminary risk assessments, technology development, obtaining permitting documentation), the main stage, the content of which is determined by the selected technology and a long monitoring stage, which makes it possible to assess the effectiveness of the taken measures. Among the main risks of in situ technology implementation, the following can be noted: the limited resources of the population that do not allow for the implementation of the procedure without prior reproduction of individuals in situ (in vitro); limited knowledge of the biology of the species; the possibility of invasion; the possibility of crossing for closely related species that сo-exist in the same habitat; social risks and consequences, target species or population may be important for the local population; financial risks during the recovery of the population. The available experience makes it possible to consider the approach to the conservation of rare species in situ as the best available technology that contributes to reducing negative environmental risks.

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