Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate ...the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergent-insoluble microdomains and significantly increased tumor necrosis factor-α-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.
Study Type – Diagnostic (exploratory cohort)
Level of Evidence 2b
What’s known on the subject? and What does the study add?
Prostate cancer surgery outcomes depend on an optimal balance of three ...aspects: complete removal of cancerous glands, preservation of nerves for sexual function and of sphincteric structures for urinary control. Current surgical techniques, even with the magnification provided by the robotic stereoscope, are insufficient to identify these structures in the surgical field. Multiphoton microscopy has been shown to produce high contrast images with subcellular resolution in fresh (unprocessed and unstained tissue) utilizing intrinsic tissue emission signals.
We provide evidence that Multiphoton microscopy of freshly excised tissue from human radical prostatectomy specimens, without any processing or use of exogenous contrast, can identify all relevant prostatic and periprostatic structures. These include the prostatic acini, the stroma and the capsule, as well as periprostatic fascial structures such as loose connective tissue, nerves, blood vessels and fat, as well as areas of local inflammation. We also show that multiphoton microscopy is able to distinguish between normal prostate gland, those with benign hyperplasia, and those harboring cancer.
OBJECTIVE
• To test whether multiphoton microscopy (MPM) might allow identification of prostatic and periprostatic structures with magnification and resolution similar to gold standard histopathology.
MATERIAL AND METHODS
• The present study included 95 robotic radical prostatectomy patients who consented to participate in an Institutional Review Board‐approved study starting in 2007.
• The types of specimens used for imaging were excised surgical margins and biopsies, and sections obtained from the excised prostate.
• The specimens were imaged with a custom‐built MPM system.
• All images were compared with haematoxylin/eosin histopathology of the same specimen.
RESULTS
• MPM of freshly excised, unprocessed and unstained tissue can identify all relevant prostatic and periprostatic structures, such as nerves, blood vessels, capsule, underlying acini and also pathological changes, including prostate cancer.
• Histological confirmation and correlation of these structures and pathologies have validated the findings of MPM.
CONCLUSIONS
• MPM shows great promise as a tool for real‐time intra‐surgical histopathology without needing excision or administration of contrast agents.
• The results will, however, need to be confirmed in true surgical settings using a miniaturized MPM microendoscope.
Objectives
To assess the ability of multiphoton microscopy (MPM) to visualise, differentiate and track periprostatic nerves in an in vivo rat model, mimicking real‐time imaging in humans during RP ...and to investigate the tissue toxicity and reproducibility of in vivo MPM on prostatic glands in the rat after imaging and final histological correlation study.
Materials and Methods
In vivo prostatic rat imaging was carried out using a custom‐built bench‐top MPM system generating real‐time three‐dimensional histological images, after performing survival surgery consisting of mini‐laparotomies under xylazine/ketamine anaesthesia exteriorising the right prostatic lobe. The acquisition time and the depth of anaesthesia were adjusted for collecting multiple images in order to track the periprostatic nerves in real‐time. The rats were then monitored for 15 days before undergoing a new set of imaging under similar settings. After humanely killing the rats, their prostates were submitted for routine histology and correlation studies.
Results
In vivo MPM images distinguished periprostatic nerves within the capsule and the prostatic glands from fresh unprocessed prostatic tissue without the use of exogenous contrast agents or biopsy sample. Real‐time nerve tracking outlining the prostate was feasible and acquisition was not disturbed by motion artefacts. No serious adverse event was reported during rat monitoring; no tissue damage due to laser was seen on the imaged lobe compared with the contralateral lobe (control) allowing comparison of their corresponding histology.
Conclusions
For the first time, we have shown that in vivo tracking of periprostatic nerves using MPM is feasible in a rat model. Development of a multiphoton endoscope for intraoperative use in humans is currently in progress and must be assessed.
Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical ...properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van‐der‐Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large‐scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non‐random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.
Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains ( e.g. DiIC 12 and FAST DiI) enter the endocytic recycling compartment efficiently, whereas ...lipid analogs with long, saturated tails ( e.g. DiIC 16 and DiIC 18 ) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F.
R. (1999) J. Cell Biol . 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result
from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate
the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties.
There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid
probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as
DiIC 16 , were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change
in overall membrane properties, and not cholesterol levels per se , by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting
enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain
a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted
cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of
intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in
contrast to the late endosomes and lysosomes.
Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol ...distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the
trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the
trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis.
Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F(v))-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, ...where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity - presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.
PDF
