Morphogenesis of Mammalian Endoplasmic Reticulum and Golgi Apparatus throughout the Cell Cycle

The endoplasmic reticulum (ER) and the Golgi apparatus are organelles that produce, modify and transport proteins and lipids and regulate Ca2+ environment within cells. Structurally they are composed of sheets and tubules. Sheets may take various forms: intact, fenestrated, single or stacked. The ER, including the nuclear envelope, is a single continuous network, while the Golgi shows only some level of connectivity. It is often unclear, how different morphologies correspond to particular functions. Previous studies indicate that the structures of the ER and Golgi are dynamic and regulated by fusion and fission events, cytoskeleton, rate of protein synthesis and secretion, and specific structural proteins. For example, many structural proteins shaping tubular ER have been identified, but sheet formation is much more unclear. In this study, we used light and electron microscopy to study morphological changes of the ER and Golgi in mammalian cells. The proportion, type, location and dynamics of ER sheets and tubules were found to vary in a cell type or cell cycle stage dependent manner. During interphase, ER and Golgi structures were demonstrated to be regulated by p37, a cofactor of the fusion factor p97, and microtubules, which also affected the localization of the organelles. Like previously shown for the Golgi, the ER displayed a tendency for fenestration and tubulation during mitosis. However, this shape change did not result in ER fragmentation as happens to Golgi, but a continuous network was retained. The activity of p97/p37 was found to be important for the reassembly of both organelles after mitosis. In EM images, ER sheet membranes appear rough, since they contain attached ribosomes, whereas tubular membranes appear smooth. Our studies revealed that structural changes of the ER towards fenestrated and tubular direction correlate with loss of ER-bound ribosomes and vice versa. High and low curvature ER membranes have a low and high density of ribosomes, respectively. To conclude, both ER and Golgi architecture depend on fusion activity of p97/p37. ER morphogenesis, particularly of the sheet shape, is intimately linked to the density of membrane bound ribosomes.Endoplasmakalvosto (ER) ja Golgi ovat organelleja, jotka tuottavat, muokkaavat ja kuljettavat proteiineja ja lipidejä sekä säätelevät kalsiumin oikeaa pitoisuutta solun sisällä. Ne koostuvat putkimaisista ja laattarakenteista, jotka voivat olla ehjiä, reikiintyneitä, yksittäisiä tai pinoutuneita. ER, johon kuuluu myös tumakalvo, on yksi yhtenäinen verkosto, kun taas Golgissa on erillisiä alaosastoja ja siis vähemmän verkostoitumista. Erilaisten rakenteiden ajatellaan tukevan erityisiä toimintoja. Useimpien rakenteiden kohdalla on kuitenkin epäselvää, mitkä toiminnot niissä sijaitsevat tai miten rakenne palvelee ko. toimintoa. Aiemmissa tutkimuksissa on todettu, että ER ja Golgi ovat dynaamisia ja niiden rakennetta muovaavat erilaiset kalvoston fuusio- ja fissiotapahtumat, solun tukiranka, proteiinituotanto ja eritys sekä rakenneproteiinit. Esimerkiksi monta putkia muodostavaa ER:n proteiinia on jo tunnistettu, mutta laattarakenteiden syntymekanismi on paljon epäselvempi. Tässä väitöskirjassa on käytetty valo- ja elektronimikroskopiaa ER:n ja Golgin rakenteiden tutkimiseen nisäkässoluissa. Eri rakenteiden määrän, laadun, paikan ja dynamiikan huomattiin vaihtelevan solutyypin mukaan. Löysimme myös näihin rakenteisiin vaikuttavan tekijän, p37:n, joka säätelee tunnettua fuusiotekijää, p97:ää. Solunjakautumisen aikana Golgi hajoaa reikiintymisen ja putkiverkoston muodostumisen kautta pieniksi rakkuloiksi ja putkiksi. Meidän tuloksemme osoittavat, että ER:ssä tapahtuu samantapaisia muodonmuutoksia, mutta ER ei hajoa palasiksi. Myös näiden muutosten laajuus vaihtelee solutyypin mukaan. P97/p37 osallistuu molempien organellien rakenteen palauttamiseen solunjakautumisen jälkeen. Elektronimikroskooppikuvissa ER:n laattarakenteet näyttävät yleensä karkealta, koska niihin on kiinnittynyt ribosomeja, jotka valmistavat proteiineja. Tutkimalla useita eri soluja, solusyklin vaiheita ja ER:n rakenteita, me havaitsimme, että ribosomien määrän väheneminen korreloi ER:n reikiintymisen tai putkiston muodostuksen tai yleensä kalvon kaarevuuden lisääntymisen kanssa. Tästä voidaan päätellä, että laattamainen ER on erikoistunut ribosomien toimintaan, josta sen muoto myös on riippuvainen

Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes

Circulating tumor DNA in early-stage breast cancer : personalized biomarkers for occult metastatic disease and risk of relapse?

The availability of blood-based markers topredict response of a solid tumor to treatment, estimate patient prognosis and diagnose relapse well before clinical symptoms arise, is a long-standing hope in clinical oncology. Ideally, assays designed to provide such information should be inexpensive (at least in the foreseeable future), simple, and, of course, predictive of the clinical evolution of the disease. While early research focused on circulating glycosylated tumor-derived protein biomarkers, the focus is now rapidly shifting to new opportunities, such as circulating tumor cells, extracellular vesicles, micro-RNAs and cancer-derived cell-free DNA a.k.a. circulating tumor-derived DNA (ctDNA).Non peer reviewe

Cancer-derived exosomes from HER2-positive cancer cells carry trastuzumab-emtansine into cancer cells leading to growth inhibition and caspase activation

Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer. The target of T-DM1 (HER2) is present also on cancer-derived exosomes. We hypothesized that exosome-bound T-DM1 may contribute to the activity of T-DM1. Methods: Exosomes were isolated from the cell culture medium of HER2-positive SKBR-3 and EFM-192A breast cancer cells, HER2-positive SNU-216 gastric cancer cells, and HER2-negative MCF-7 breast cancer cells by serial centrifugations including two ultracentrifugations, and treated with T-DM1. T-DM1 not bound to exosomes was removed using HER2-coated magnetic beads. Exosome samples were analyzed by electron microscopy, flow cytometry and Western blotting. Binding of T-DM1-containing exosomes to cancer cells and T-DM1 internalization were investigated with confocal microscopy. Effects of T-DM1-containg exosomes on cancer cells were investigated with the AlamarBlue cell proliferation assay and the Caspase-Glo 3/7 caspase activation assay. Results: T-DM1 binds to exosomes derived from HER2-positive cancer cells, but not to exosomes derived from HER2-negative MCF-7 cells. HER2-positive SKBR-3 cells accumulated T-DM1 after being treated with T-DM1-containg exosomes, and treatment of SKBR-3 and EFM-192A cells with T-DM1-containing exosomes resulted in growth inhibition and activation of caspases 3 and/or 7. Conclusion: T-DM1 binds to exosomes derived from HER2-positive cancer cells, and T-DM1 may be carried to other cancer cells via exosomes leading to reduced viability of the recipient cells. The results suggest a new mechanism of action for T-DM1, mediated by exosomes derived from HER2-positive cancer.Peer reviewe

Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells

The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes

Exploration of Extracellular Vesicle miRNAs, Targeted mRNAs and Pathways in Prostate Cancer : Relation to Disease Status and Progression

Simple Summary Prostate cancer lacks non-invasive specific biomarkers for aggressive disease. Urinary extracellular vesicles (uEV) could provide such markers; however, due to technical challenges, little is known regarding the pathogenesis pathways reflected in uEV. We performed a miRNA, target mRNA and pathway study focused on uEV, exploring the differences between cancer (1) status groups (Gleason score) and (2) progression groups. The uEV provided a surprisingly comprehensive presentation of differentially expressed miRNAs, target mRNAs and pathogenesis pathways. The miRNAs associated with prostate cancer status or progression were mostly unique, but still targeted overlapping sets of signalling, resistance, hormonal and immune pathways. Interestingly, mRNA targets of the key miRNAs (miR-892a, miR-223-3p, miR-146a-5p) were widely expressed in both uEV and plasma EV from PCa patients. The study thus suggests that uEV carry a vast presentation of PCa status and progression-linked RNAs that are worth further exploration in large personalized medicine trials. Background: Prostate cancer (PCa) lacks non-invasive specific biomarkers for aggressive disease. We studied the potential of urinary extracellular vesicles (uEV) as a liquid PCa biopsy by focusing on the micro RNA (miRNA) cargo, target messenger RNA (mRNA) and pathway analysis. Methods: We subjected uEV samples from 31 PCa patients (pre-prostatectomy) to miRNA sequencing and matched uEV and plasma EV (pEV) from three PCa patients to mRNA sequencing. EV quality control was performed by electron microscopy, Western blotting and particle and RNA analysis. We compared miRNA expression based on PCa status (Gleason Score) and progression (post-prostatectomy follow-up) and confirmed selected miRNAs by quantitative PCR. Expression of target mRNAs was mapped in matched EV. Results: Quality control showed typical small uEV, pEV, RNA and EV-protein marker enriched samples. Comparisons between PCa groups revealed mostly unique differentially expressed miRNAs. However, they targeted comprehensive and largely overlapping sets of cancer and progression-associated signalling, resistance, hormonal and immune pathways. Quantitative PCR confirmed changes in miR-892a (Gleason Score 7 vs. >= 8), miR-223-3p (progression vs. no progression) and miR-146a-5p (both comparisons). Their target mRNAs were expressed widely in PCa EV. Conclusions: PCa status and progression-linked RNAs in uEV are worth exploration in large personalized medicine trials.Peer reviewe

Exploration of Extracellular Vesicle miRNAs, Targeted mRNAs and Pathways in Prostate Cancer: Relation to Disease Status and Progression

Background: Prostate cancer (PCa) lacks non-invasive specific biomarkers for aggressive disease. We studied the potential of urinary extracellular vesicles (uEV) as a liquid PCa biopsy by focusing on the micro RNA (miRNA) cargo, target messenger RNA (mRNA) and pathway analysis. Methods: We subjected uEV samples from 31 PCa patients (pre-prostatectomy) to miRNA sequencing and matched uEV and plasma EV (pEV) from three PCa patients to mRNA sequencing. EV quality control was performed by electron microscopy, Western blotting and particle and RNA analysis. We compared miRNA expression based on PCa status (Gleason Score) and progression (post-prostatectomy follow-up) and confirmed selected miRNAs by quantitative PCR. Expression of target mRNAs was mapped in matched EV. Results: Quality control showed typical small uEV, pEV, RNA and EV-protein marker enriched samples. Comparisons between PCa groups revealed mostly unique differentially expressed miRNAs. However, they targeted comprehensive and largely overlapping sets of cancer and progression-associated signalling, resistance, hormonal and immune pathways. Quantitative PCR confirmed changes in miR-892a (Gleason Score 7 vs. ≥8), miR-223-3p (progression vs. no progression) and miR-146a-5p (both comparisons). Their target mRNAs were expressed widely in PCa EV. Conclusions: PCa status and progression-linked RNAs in uEV are worth exploration in large personalized medicine trials

Exploration of Extracellular Vesicle miRNAs, Targeted mRNAs and Pathways in Prostate Cancer : Relation to Disease Status and Progression

Simple Summary Prostate cancer lacks non-invasive specific biomarkers for aggressive disease. Urinary extracellular vesicles (uEV) could provide such markers; however, due to technical challenges, little is known regarding the pathogenesis pathways reflected in uEV. We performed a miRNA, target mRNA and pathway study focused on uEV, exploring the differences between cancer (1) status groups (Gleason score) and (2) progression groups. The uEV provided a surprisingly comprehensive presentation of differentially expressed miRNAs, target mRNAs and pathogenesis pathways. The miRNAs associated with prostate cancer status or progression were mostly unique, but still targeted overlapping sets of signalling, resistance, hormonal and immune pathways. Interestingly, mRNA targets of the key miRNAs (miR-892a, miR-223-3p, miR-146a-5p) were widely expressed in both uEV and plasma EV from PCa patients. The study thus suggests that uEV carry a vast presentation of PCa status and progression-linked RNAs that are worth further exploration in large personalized medicine trials. Background: Prostate cancer (PCa) lacks non-invasive specific biomarkers for aggressive disease. We studied the potential of urinary extracellular vesicles (uEV) as a liquid PCa biopsy by focusing on the micro RNA (miRNA) cargo, target messenger RNA (mRNA) and pathway analysis. Methods: We subjected uEV samples from 31 PCa patients (pre-prostatectomy) to miRNA sequencing and matched uEV and plasma EV (pEV) from three PCa patients to mRNA sequencing. EV quality control was performed by electron microscopy, Western blotting and particle and RNA analysis. We compared miRNA expression based on PCa status (Gleason Score) and progression (post-prostatectomy follow-up) and confirmed selected miRNAs by quantitative PCR. Expression of target mRNAs was mapped in matched EV. Results: Quality control showed typical small uEV, pEV, RNA and EV-protein marker enriched samples. Comparisons between PCa groups revealed mostly unique differentially expressed miRNAs. However, they targeted comprehensive and largely overlapping sets of cancer and progression-associated signalling, resistance, hormonal and immune pathways. Quantitative PCR confirmed changes in miR-892a (Gleason Score 7 vs. >= 8), miR-223-3p (progression vs. no progression) and miR-146a-5p (both comparisons). Their target mRNAs were expressed widely in PCa EV. Conclusions: PCa status and progression-linked RNAs in uEV are worth exploration in large personalized medicine trials.Peer reviewe

Urinary extracellular vesicles carry multiple activators and regulators of coagulation

Cells shape their extracellular milieu by secreting intracellular products into the environment including extracellular vesicles which are lipid-bilayer limited membrane particles. These vesicles carry out a range of functions, including regulation of coagulation, via multiple contributor mechanisms. Urinary extracellular vesicles are secreted by various cells, lining the urinary space, including the nephron and bladder. They are known to have procoagulant properties, however, the details of this function, beyond tissue factor are not well known. The aim of the study was to access the role of urinary extracellular vesicles in impacting coagulation upon supplementation to plasma. This could indicate their physiological function upon kidney injury or pathology. Supplementation to standard human plasma and plasmas deficient in various coagulation factors was used for this purpose, and calibrated automated thrombogram (CAT (R)) was the major technique applied. We found that these vesicles contain multiple coagulation-related factors, and their lipid composition affects coagulation activities of plasma upon supplementation. Remarkably, these vesicles can restore thrombin generation in FVII, FVIII, FIX and FXI -deficient plasmas. This study explores the multiple roles of urinary extracellular vesicles in coagulation in in vitro blood coagulation and implies their importance in its regulation by several mechanisms.Peer reviewe

Efficient ultrafiltration based protocol to deplete extracellular vesicles from fetal bovine serum

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EVdepleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.Peer reviewe