Merging complexes: properties of membrane raft assembly during lymphocyte signaling.
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Animals↗
Biomedical subjects
William Rodgers
Publications and source records attributed to William Rodgers.
Properties of glycolipid-enriched membrane rafts in antigen presentation.
Presentation of antigen to T cells represents one of the central events in the engagement of the immune system toward the defense of the host against pathogens. Accordingly, understanding the mechanisms by which antigen presentation occurs is critical toward our understanding the properties of host defense against foreign antigen, as well as insight into other features of the immune system, such as autoimmune disease. The entire antigen-presentation event is complex, and many features of it remain poorly understood. However, recent studies have provided evidence showing that glycolipid-enriched membrane rafts are important for efficient antigen presentation; the studies suggest that one such function of rafts is trafficking of antigen-MHC II complexes to the presentation site on the surface of the antigen-presenting cell. Here, we present a critical discussion of rafts and their proposed functions in antigen presentation. Emerging topics of rafts and antigen presentation that warrant further investigation are also highlighted.
Actins↗
Proteomic profiling of mature CD10+ B-cell lymphomas.
Proteomic profiling with protein-chip technology has been used successfully to discover biomarkers with potential clinical usefulness in several cancer types. Little proteomic study has been done in B-cell lymphomas. We determined whether the expression of a set of proteins by protein-chip technology coupled with new informatics tools could be used to build a model to molecularly classify B-cell lymphoma subgroups. We used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze 18 CD10+ B-cell lymphomas, including 6 grade 1 (G1) follicular lymphomas (FLs), 7 grade 3 (G3) FLs, and 5 Burkitt lymphomas. We used 7 reactive follicular hyperplasia cases as a control group. By using SAX2 ProteinChip arrays (Ciphergen Biosystems, Fremont, CA), we found a unique protein expression profile for each type of lesion. Two-way hierarchical clustering analysis of these protein expression profiles differentiated reactive follicular hyperplasia, FL, and Burkitt lymphoma, with 5 major clusters of differentially expressed protein peaks. In addition, we identified histone H4 as a potential differentially expressed protein marker that seems to distinguish G1 from G3 FL. To our knowledge, this is the first proteomic study using protein-chip technology for molecular classification of B-cell lymphoma subtypes with clinical samples.
Biomarkers, Tumor↗
Visualization of transfer of a fluorescently-labeled membrane raft protein to T cells using lentivirus.
Lentivirus vector systems have been developed for the safe delivery of foreign genes to target tissues. However, the use of these systems for delivering specific proteins to target cells has been largely unexplored. To test this concept, the lentivirus expression plasmid pLenti was utilized to overexpress in producer cells a YFP-fusion protein that is specifically targeted to glycolipid-enriched membrane rafts, which is the site of virus assembly. Our data show that virus generated in producer cells that expressed the YFP fusion protein were able to effectively label target cells by a 2-3 hr incubation with the virus. Labeling of the target cells was specific to the lentivirus, as it was blocked by pre-incubating the virus with antibody to the surface protein, and it was not affected by pre-treating the target cells with cyclohexamide. T cells that were labeled using the lentivirus underwent a robust stimulation following crosslinking the T cell receptor, thus showing that T cells labeled using lentivirus remained responsive to extracellular cues. Altogether, these results show that overexpression of foreign proteins in lentivirus producer cells can yield protein-loaded viruses, which can then function to deliver the protein to target cells. Thus, our findings suggest an avenue for targeting specific proteins to cells where foreign gene expression is not feasible.
Journal Article↗
Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA-PK cofactor InsP6 inhibits Ku mobility.
Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA-PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP6) is a potent cofactor for DNA-PK activity in NHEJ. Specifically, InsP6 binds to the Ku component of DNA-PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP6 on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku's interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP6 was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku-GFP, and this could be reversed by replenishing cells with InsP6. By expressing deletion mutants of Ku-GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins.
Antigens, Nuclear↗
Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs.
Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.
Actins↗
Prospective comparison of brush cytology, viral culture, and histology for the diagnosis of ulcerative esophagitis in AIDS.
BACKGROUND & AIMS: The best method(s) for the diagnosis of esophageal ulcers in patients with acquired immune deficiency syndrome (AIDS) is unknown. METHODS: Over an 8-year period, all human immunodeficiency virus (HIV)-infected patients with a CD4 lymphocyte count of < or =250/mm(3) and an esophageal ulcer underwent a standard protocol consisting of 2 biopsy examinations for viral culture (shell vial technique), brushing for cytology with immunohistochemistry (IHC) for viral antigens, and 10 biopsy examinations of the ulcer base/edge for routine histology and IHC. The cause of ulcer was based on histologic findings and clinical and endoscopic follow-up evaluation after therapy. RESULTS: Forty patients with 48 episodes of ulcer were studied (mean age, 34 yr; 88% men; median CD4 lymphocyte count = 31 cells/mm(3); range, 0-250 cells/mm(3)). Six patients had more than one endoscopic examination documenting ulcer, and 2 patients had more than one cause of ulcer. The most common causes of ulcer were: idiopathic (IEU) 22, cytomegalovirus (CMV) 15, and gastroesophageal reflux disease 6. Viral culture was positive for herpes simplex in 3 of 3, but only 4 of 15 (27%) for CMV, and specificity of viral culture was 100%. Cytologic brushings were only positive in the patients with herpes simplex virus (HSV) or incidental Candida esophagitis. Viral cytopathic effect was apparent on routine histologic staining in each patient with viral esophagitis, and IHC confirmed the diagnosis in each patient. Follow-up evaluation disclosed no patient with a misdiagnosis based on histology. CONCLUSIONS: Viral culture and cytologic brushings add little to the evaluation of esophageal ulcers in patients with AIDS over multiple biopsy specimens with routine histology.
Acquired Immunodeficiency Syndrome↗
T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses.
In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.
Actins↗
Transient association of Ku with nuclear substrates characterized using fluorescence photobleaching.
The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining. Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes. Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates. To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86. The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells. However, the rate of diffusion of Ku was approximately 100-fold slower than that predicted from its size. Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix. A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix. These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold.
Active Transport, Cell Nucleus↗
Surgical staging in patients with ovarian tumors of low malignant potential.
OBJECTIVE: To compare the outcomes of patients with ovarian tumors of low malignant potential who had complete surgical staging with those who were unstaged to determine whether the rate of recurrence or survival was affected by surgical staging. METHODS: A retrospective chart review was performed on 93 consecutive patients who had surgery for histologically confirmed tumors of low malignant potential between 1979 and 1997. Two cohorts of patients were identified: patients who had classic surgical staging (n = 48) versus those who were not staged (n = 45). Outcome data were recorded for patients and compared between the two groups. RESULTS: Early stage (I or II) disease was diagnosed in 31 of 48 patients who had surgical staging and 42 of 45 patients who were not staged (P =.001). In 17% of patients their stage was upgraded on the basis of surgical staging, as a result of retroperitoneal involvement in only 6% of those cases (three of 48 staged patients). During the study interval, the frozen section diagnosis of low malignant potential tumor of the ovary was changed to a final diagnosis of invasive cancer in eight other patients. There were three recurrences and two deaths in both the staged and unstaged low malignant potential groups. The average duration of follow-up was 6.5 +/- 4.2 years and was similar in the two groups. Overall 5-year survival was approximately 93% for all stages. CONCLUSION: Survival and recurrence rates were not significantly different between staged and unstaged patients who had surgery for low malignant potential tumors of the ovary.
Adenocarcinoma, Mucinous↗
Automated method for quantifying fluorophore colocalization in fluorescence double-labeling experiments.
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Automation↗
Making membranes green: construction and characterization of GFP-fusion proteins targeted to discrete plasma membrane domains.
Cell membranes contain glycolipid-enriched membrane (GEM) domains, or lipid rafts. GEM domains represent a discrete assembly ofproteins and lipids within the plasma membrane thatfunctions in cell signaling. However, studies of the GEM domains often include the disruption of cells with detergent. Thus, many of the physical and biological properties of GEM domains remain unknown and even controversial. An approach to study these domains but avoid detergent lysis is to measure their properties using the fluorescence imaging of live cells. Accordingly, GFP was targeted to either the GEM or the non-GEMfraction of the plasma membrane using the minimal membrane-anchoring signals of p56lck and pp60c-Src, respectively. The targeting of the fusion proteins to the respective membrane fractions was assayed by membrane fractionation and by quantitating the enrichment in GEM caps in stimulated T cells. The results show that the GEM marker was targeted to GEM domains with similar efficiency as other GEM-associated proteins. Conversely, the non-GEM marker was completely excluded from GEM domains. These constructs represent a useful toolfor studying the discrete fractions of the plasma membrane in live cells using fluorescence imaging.
Cell Membrane↗
Unusual fallopian tube neoplasm in a 10-year-old patient with Proteus syndrome: a case report and review of the literature.
We report a case of fallopian tube and peritoneal lesions resected from a 10-year-old girl with Proteus syndrome. The papillary fallopian tube tumor expressed CA-125 and demonstrated negative staining for CEA, CD15, calretinin, and CK 5/6. Nondilated portions of fallopian tube exhibited evidence of an in situ component. This patient was also found to have peritoneal mesothelial hyperplasia as well as cystic peritoneal lesions of mesothelial origin, with positive calretinin staining. This case provides direct evidence of both Müllerian and mesothelial lesions arising concurrently in a patient with Proteus syndrome and represents the 1st documented fallopian tube neoplasm. Previously reported lesions involving the female and male genital tracts in patients with Proteus syndrome are reviewed.
Biomarkers, Tumor↗