scholarly journals A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

2015 ◽  
Vol 208 (3) ◽  
pp. 273-281 ◽  
Author(s):  
Marvin Bentley ◽  
Helena Decker ◽  
Julie Luisi ◽  
Gary Banker
Keyword(s):  
Experimental Evidence ◽  
Convenient Method ◽  
Cell Biology ◽  
Fundamental Problem ◽  
Family Members ◽  
Binding Domain ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Preferential Binding ◽  
Candidate Protein

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations.

Gene-based Therapeutic Tools in the Treatment of Cornea Disease

2019 ◽  
Vol 19 (1) ◽  
pp. 7-19 ◽  
Author(s):  
Xiao-Xiao Lu ◽  
Shao-Zhen Zhao
Keyword(s):  
Protein Expression ◽  
Cell Biology ◽  
Inflammatory Cells ◽  
Vector System ◽  
Corneal Disease ◽  
Corneal Injury ◽  
Transfer Vectors ◽  
Pathological Development ◽  
Candidate Protein ◽  
Therapeutic Tools

Background: As one of the main blinding ocular diseases, corneal blindness resulted from neovascularization that disrupts the angiogenic privilege of corneal avascularity. Following neovascularization, inflammatory cells are infiltrating into cornea to strengthen corneal injury. How to maintain corneal angiogenic privilege to treat corneal disease has been investigated for decades. Methodology: Local administration of viral and non-viral-mediated anti-angiogenic factors reduces angiogenic protein expression in situ with limited or free of off-target effects upon gene delivery. Recently, Mesenchymal Stem Cells (MSCs) have been studied to treat corneal diseases. Once MSCs are manipulated to express certain genes of interest, they could achieve superior therapeutic efficacy after transplantation. Discussion: In the text, we first introduce the pathological development of corneal disease in the aspects of neovascularization and inflammation. We summarize how MSCs become an ideal candidate in cell therapy for treating injured cornea, focusing on cell biology, property and features. We provide an updated review of gene-based therapies in animals and preclinical studies in the aspects of controlling target gene expression, safety and efficacy. Gene transfer vectors are potent to induce candidate protein expression. Delivered by vectors, MSCs are equipped with certain characters by expressing a protein of interest, which facilitates better for MSC-mediated therapeutic intervention for the treatment of corneal disease. Conclusion: As the core of this review, we discuss how MSCs could be engineered to be vector system to achieve enhanced therapeutic efficiency after injection.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

Assessment of medication intake forgetfulness, recent digital medication technology and associated individuals’ acceptance and health privacy concerns

2021 ◽  
pp. 4934-4940
Author(s):  
Sabrina Ait Gacem ◽  
Nageeb AGM Hassan ◽  
Afnan Abdul-Hameed Al-Qaysi ◽  
Maryam Jaafar AlAani
Keyword(s):  
Mobile Application ◽  
Fundamental Problem ◽  
Statistical Tests ◽  
New Technology ◽  
Family Members ◽  
Vital Role ◽  
Hard Copy ◽  
Cross Sectional ◽  
Adherence To Medication ◽  
Study Results

Background: Non-adherence to medication is a fundamental problem worldwide that leads to further complications. In corresponding to this problem, The FDA approved a drug with an embedded sensor, that sends signals through the Bluetooth to the application and website and this will play a vital role in improving the adherence, as it shows whether the drug has been ingested or not. Objectives: The purpose of this study is to investigate the importance of adherence, evaluating of respondents' impression toward the new technologist medication "Digital Pill" that will be applied in the future for a range of medications, as well as discussing patients’ opinions regarding privacy issues related to digital pills use. Methods: This was a cross-sectional study involving 320 university students during the period of one month from June to July 2018. The data was obtained through hard copy and online (electronic) survey. The data was analysed using SPSS version 20. Results: The current study results show that (61.9%) of respondents sometimes forget to take their medication followed by (24.4%, 13.8%) who never/rarely and usually/always forget to take their medications, respectively. Surprisingly, the majority of respondents (35%) take their medication anyway when they forgot to take it and (33.4%) they skip the dose of the medication and only few of respondents (28.1%) stated that they ask family members to know what to do when they forget to take the medication. More than half of the respondents (53.4%) do not tell their doctor if they forgot to take the medication. Majority of respondents (44.7%) ask family members to remind them to take their medication. Approximately two-thirds of the respondents (61.3%) said that they stopped taking their medication without telling the doctor. Further statistical tests revealed that most respondents (78.8%) want to use the "Digital pill". On average, (55.9%) of respondents were shown a full agreement to allow the doctor to access their mobile application and website. Conclusion: Based on the conducted study, we conclude that most of the respondents gave positive and good feedback and agreed to use such a new technology "Digital Pill" as they found it very helpful and will lead to improved health outcomes. As well as they also agreed to allow the doctor to access their mobile application and website to check if they take medication or not.

Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network

1999 ◽  
Vol 112 (6) ◽  
pp. 845-854 ◽  
Author(s):  
A.C. Valdez ◽  
J.P. Cabaniols ◽  
M.J. Brown ◽  
P.A. Roche
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Family Members ◽  
Snare Proteins ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Syntaxin 11 ◽  
Golgi Network

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

scholarly journals The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors

2020 ◽  
Vol 21 (24) ◽  
pp. 9352
Author(s):  
Manh Tien Tran ◽  
Yuka Okusha ◽  
Yunxia Feng ◽  
Masatoshi Morimatsu ◽  
Penggong Wei ◽  
...  
Keyword(s):  
Osteoclast Differentiation ◽  
Activated T Cells ◽  
Surface Receptors ◽  
Late Endosomes ◽  
Early Endosomes ◽  
D Cell ◽  
D Cells ◽  
Vitro Model

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals Sorting to Synaptic-like Microvesicles from Early and Late Endosomes Requires Overlapping but Not Identical Targeting Signals

2000 ◽  
Vol 11 (5) ◽  
pp. 1801-1814 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Daniel F. Cutler
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Epidermal Growth

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.

scholarly journals Internalization and endosomal degradation of receptor-bound antigens regulate the efficiency of cross presentation by human dendritic cells

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2011-2020 ◽  
Author(s):  
Bithi Chatterjee ◽  
Anna Smed-Sörensen ◽  
Lillian Cohn ◽  
Cécile Chalouni ◽  
Richard Vandlen ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Vaccine Development ◽  
Mannose Receptor ◽  
Antigen Delivery ◽  
Cross Presentation ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Antibody Conjugates ◽  
Human Dendritic Cells ◽  
Endocytic Compartments

Abstract Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.

Sign in / Sign up

Export Citation Format

Share Document