Journal articles on the topic 'Organisation subcellulaire'

Numb is an adaptor protein that controls the fate of cells in different species through asymmetrical inheritance by sibling cells during division. It has been investigated extensively in mitosis, mostly in neural progenitor cells, but its function in meiosis remains unknown. The present study was designed to investigate the expression, subcellular localisation and functional roles of Numb during mouse oocyte meiotic maturation. Using real-time polymerase chain reaction and western blotting, we found that the expression of Numb increased from the germinal vesicle (GV) to MII stages. Immunofluorescent staining revealed that Numb was mainly concentrated in the GV before meiosis resumption, aggregated in the vicinity of the chromosomes after GV breakdown and then localised to the spindle poles from prometaphase I to MII. Nocodazole treatment resulted in spindle destruction and Numb diffusion into the cytoplasm. However, Numb appeared at the spindle poles again once the spindles had formed when nocodazole-treated oocytes were washed and cultured for spindle recovery. Depletion of Numb by RNA interference resulted in chromosome misalignment, spindle deformation and even doubled spindle formation. Our results suggest that Numb is critical for spindle organisation during mouse oocytes meiosis. The present study provides evidence of a new function for Numb in addition to its action as a cell fate-determining factor.

The recent increase in use of Field Emission Scanning Instruments in biology has shown the potential of these systems for sub-cellular imaging, as they essentially provide transmission EM levels of resolution for surface imaging. in structures lacking inherent contrast in thin sections, such as nuclear pore complexes,(NPC) high resolution surface imaging has provided significant complementary structural information to the macromolecular architecture of the NPC. (Goldberg and Allen, 1996). A series of intermediate structural forms of the NPC during its formation has also been described using FESEM, which would not have been resolvable in TEM.(Goldberg, Weise, Allen, Wilson, 1997). All this work was performed with chemical fixation, dehydration and critical point drying, followed by high resolution Chromium coating. We now are extending these studies to a cryo-preservation approach, mindful of the requirement of rapid freezing to preserve the more labile events of membrane dynamics, and also to circumvent the requirement to expose membranes to organic solvents during conventional dehydration.

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

AbstractThe female reproductive system of the free-living nematode family Cephalobidae is examined by means of differential interference contrast, scanning electron and fluorescent microscopy. The model nematode Caenorhabditis elegans and the predatory nematode Prionchulus punctatus are also included in this study; the former mainly to test our results with the very detailed knowledge of this model organism, the latter to provide a representative of the more distantly related Enoplea. In this comparative approach, the analysed gonad structures are discussed with respect to their functional and phylogenetic significance. The general cellular composition of the cephalobid gonad – namely an oviduct comprising two rows of four cells, a distinctly offset spermatheca consisting of 8-16 cells, and a uterus composed of distinct cell rows – differs from all known Nematoda except that of the plant-parasitic Tylenchomorpha. Despite the striking evolutionary conservation of the cellular architecture of the cephalobid gonad there is a complex subcellular specialisation, namely a significant and functionally relevant variation in myofilament organisation, both among Cephalobidae and between major groups of nematodes. We demonstrate the presence of microfilaments that vary in pattern among species and that may play an important role in egg propulsion. The phenomenon of endotokia matricida, in which eggs do not leave the female body, is found to be associated with a massive rupture of the cytoskeleton in the uterus wall. The complexity of the myofibril structure and the associated potential to propagate oocytes actively cannot be solely explained by differences in phylogenetic history, but is also linked to body diameter. In the larger Acrobeloides maximus, the proximal end of the ovary sheath is adorned with 12 distinct longitudinal bands, antibody binding positively for paramyosin, while in the smaller Cephalobus cubaensis myofilament organisation is at random.

LIM kinase 1 (LIMK1) activity is essential for cell migration and cell cycle progression. Little is known about LIMK1 expression and function in mammalian oocytes. In the present study we assessed LIMK1 protein expression, subcellular distribution and function during mouse oocyte meiosis. Western blot analysis revealed high and stable expression of LIMK1 from the germinal vesicle (GV) to MII stage. In contrast, activated LIMK1 (i.e. LIMK1 phosphorylated at threonine 508 (pLIMK1Thr508)) was only detected after GV breakdown, with levels increasing gradually to peak at MI and MII. Immunofluorescence showed pLIMK1Thr508 was colocalised with the microtubule organising centre (MTOC) components pericentrin and γ-tubulin at the spindle poles. A direct interaction between γ-tubulin and pLIMK1Thr508 was confirmed by co-immunoprecipitation. LIMK inhibition with 1 μM BMS3 damaged MTOC protein localisation to spindle poles, undermined the formation and positioning of functional MTOC and thus disrupted spindle formation and chromosome alignment. These effects were phenocopied by microinjection of LIMK1 antibody into mouse oocytes. In summary, the data demonstrate that LIMK activity is essential for MTOC organisation and distribution and so bipolar spindle formation and maintenance in mouse oocytes.

Ryanodine receptors (RyRs) are expressed on the endoplasmic reticulum of many cells, where they form intracellular Ca2+-release channels that participate in the generation of intracellular Ca2+ signals. Here we report studies on the intracellular localisation and functional properties of transfected RyR1 or RyR3 channels in HEK 293 cells. Immunofluorescence studies indicated that both RyR1 and RyR3 did not form clusters but were homogeneously distributed throughout the endoplasmic reticulum. Ca2+ release experiments showed that transfected RyR1 and RyR3 channels responded to caffeine, although with different sensitivity,generating a global release of Ca2+ from the entire endoplasmic reticulum. However, video imaging and confocal microscopy analysis revealed that, in RyR3-expressing cells, local spontaneous Ca2+ release events were observed. No such spontaneous activity was observed in RyR1-expressing cells or in control cells. Interestingly, the spontaneous release events observed in RyR3-expressing cells were restricted to one or two regions of the endoplasmic reticulum, suggesting the formation of a further subcellular organisation of RyR3 in Ca2+ release units. These results demonstrate that different RyR isoforms can engage in the generation of distinct intracellular Ca2+ signals in HEK 293 cells.

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus–oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB−(colourless cytoplasm, high G6PDH activity) and BCB+(coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB+and BCB−oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB+(33.1±3.1%) and BCB−(12.1±1.5%) oocytes. Moreover, BCB+oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein,NASP) and protein biosynthesis (RPS274Aand mRNA for elongation factor 1α,EF1A). BCB−oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15,BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.

Nitrogen-fixing root nodules are plant organs specialised for symbiotic transfer of nitrogen and carbon between microsymbiont and host. The organisation of nitrogen assimilation, storage and transport processes is partitioned at the subcellular and tissue levels, in distinctive patterns depending on the symbiotic partners. In this review, recent advances in understanding of actinorhizal nodule nitrogen assimilation are presented. New findings indicate that Frankia within nodules of Datisca glomerata (Presl.) Baill. carries out both primary nitrogen assimilation and biosynthesis of arginine, rather than exporting ammonium. Arginine is a typical storage form of nitrogen in plant tissues, but is a novel nitrogen carrier molecule in root nodule symbioses. Thus Frankia within D. glomerata nodules exhibits considerable metabolic independence. Furthermore, nitrogen reassimilation is likely to take place in the host in the uninfected nodule cortical cells of this root nodule symbiosis, before amino acid export to host sink tissues via the xylem. The role of an augmented pericycle in carbon and nitrogen exchange in root nodules deserves further attention in actinorhizal symbiosis, and further highlights the importance of a comprehensive, structure–function approach to understanding function in root nodules. Moreover, the multiple patterns of compartmentalisation in relation to nitrogen flux within root nodules demonstrate the diversity of possible functional interactions between host and microsymbiont that have evolved in the nitrogen-fixing clade.

p21-activated kinase 1 (Pak1) is essential for a variety of cellular events, including gene transcription, cytoskeletal organisation, cell proliferation and apoptosis. Pak1 is activated upon autophosphorylation on many amino residues; in particular, phosphorylation on Thr423 maintains maximal Pak1 activation. In the present study we investigated the protein expression, subcellular localisation and function of Pak1 phosphorylated on Thr423 (pPak1Thr423) in mouse oocytes. pPak1Thr423 was detected upon meiotic resumption and localised on the condensing chromatin. Thr423 phosphorylation was markedly suppressed by the Pak1 ATP-competitive inhibitor PF-3758309, but not by the allosteric inhibitors IPA-3 (2.5 μM and 10 μM) (1, 1′-dithiobis-2-naphthalenol) and TAT-PAK18 (10 μM), which prevent the binding of Pak1 to its upstream activators GTPase Cdc42/Rac and Pak-interacting exchange factor (PIX), respectively, implying that Pak1 activation may be independent of GTPase and PIX in oocyte meiosis. Inhibition of Pak1 activation concomitantly restrained histone H3 phosphorylation on Ser10 and consequently inhibited chromatin condensation; however, this phenotype was reversed by concomitant administration of the Pak1 activator FTY720. The changes in the pattern of expression of phosphorylated extracellular signal-regulated kinase 1/2 in response to PF-3758309 or FTY720 were the same as seen for pPak1Thr423. These results show that activated Pak1 regulates chromatin condensation by promoting H3 Ser10 phosphorylation in oocytes after the resumption of meiotic progression.

The process of nitrate assimilation is a very crucial pathway for the sustainable growth and productivity of higher plants. This process is catalysed by two enzymes, nitrate reductase and nitrite reductase. Both the enzymes differ from each other with respect to their structural organisation, subcellular location, catalytic efficiencies and regulatory mechanisms. Nitrate reductase catalyses the rate limiting step of nitrate assimilation process. The genes and proteins of this enzyme have been isolated and characterised from many higher plants. The additional role of NR in the production of nitric oxide has been also reported in last several years. The reduced ammonium is assimilated into carbon skeleton, ?-ketoglutarate, by the concerted action of glutamine synthetase and glutamate synthase. Glutamine and glutamate are the transportable forms of nitrogen among various tissues and metabolic processes. The rate of nitrate assimilation is regulated by the rate of uptake of nitrate by nitrate transporters, availability of carbon skeleton, accumulation of nitrogenous end products, light and the rate of photosynthesis. The partitioning of metabolites and resources between carbon and nitrogen metabolism is an important factor for the growth and yield of plants. During the last several decades excess use of nitrogen fertiliser has caused environmental pollution. Efforts have been made to increase the nitrogen use efficiency of plants to reduce the cost on fertiliser and nitrate pollution, increase the productivity and protein content of several commonly used crops. This review discusses the process of nitrate assimilation and its interaction with the carbon metabolism.

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor–phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor–phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

AbstractDuchenne muscular dystrophy (DMD) is caused by frameshift mutations in the DMD gene that prevent the body-wide translation of its protein product, dystrophin. Besides a severe muscle phenotype, cognitive impairment and neuropsychiatric symptoms are prevalent. Dystrophin protein 71 (Dp71) is the major DMD gene product expressed in the brain and mutations affecting its expression are associated with the DMD neuropsychiatric syndrome. As with dystrophin in muscle, Dp71 localises to dystrophin-associated protein complexes in the brain. However, unlike in skeletal muscle; in the brain, Dp71 is alternatively spliced to produce many isoforms with differential subcellular localisations and diverse cellular functions. These include neuronal differentiation, adhesion, cell division and excitatory synapse organisation as well as nuclear functions such as nuclear scaffolding and DNA repair. In this review, we first describe brain involvement in DMD and the abnormalities observed in the DMD brain. We then review the gene expression, RNA processing and functions of Dp71. We review genotype-phenotype correlations and discuss emerging cellular/tissue evidence for the involvement of Dp71 in the neuropathophysiology of DMD. The literature suggests changes observed in the DMD brain are neurodevelopmental in origin and that their risk and severity is associated with a cumulative loss of distal DMD gene products such as Dp71. The high risk of neuropsychiatric syndromes in Duchenne patients warrants early intervention to achieve the best possible quality of life. Unravelling the function and pathophysiological significance of dystrophin in the brain has become a high research priority to inform the development of brain-targeting treatments for Duchenne.

Abstract The thyroid is essential for maintaining systemic homeostasis by regulating thyroid hormone concentrations in the bloodstream. Due to the limited number of representative model systems, there is limited understanding of fundamental thyroid biology as well as thyroid carcinogenesis. To fill the caveats in the understanding of thyroid cell biology, we aimed to develop an adult stem cell-derived three-dimensional (3D) organoid culture system using murine and human thyroid follicular cells (TFCs). We have succeeded to grow such an organoid culture system that harbours the complete machinery of hormone production visualised by the presence of colloid in the lumen and essential transporters and enzymes in a polarised cell layer. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1/TTF1. Moreover, the thyroid hormone precursor thyroglobulin is expressed in both cultures to similar levels as in tissue. Extensive characterisation furthermore identifies known and new biological insights in TFC subclassification, subcellular organisation and hormone production using state-of-the art techniques like single cell RNA sequencing, transmission electron microscopy and genome editing. These 3D in vitro cultures allow for a variety of thyroid-related studies including the progression of wild type cells towards cancer. Additionally, due to the success of generating patient-specific tumour organoids of primary differentiated thyroid carcinoma and metastasis, insights in drug resistance and metastases can be identified. In short, this newly developed organoid culture of murine and human wild type TFCs as well as tumour tissue opens up an extensive area of research that will help understand the drivers for growth and development of thyroid (cancer) cells and enable studies upon drug responsiveness.

In vitro plant cells in culture release proteins and carbohydrates, but the active molecules responsible for sustaining the switch in embryogenic development and progression have not yet been identified. In maize (Zea mays L.), the Esr genes encode for small hydrophilic proteins and are expressed in the restricted region of maize endosperm surrounding the embryo: the embryo surrounding region (ESR). In the present work, the possible influence of secreted molecules in the liquid medium during microspore-derived embryo development and specifically, the presence of Esr proteins, has been analysed in maize microspore cultures. The study has been conducted with in situ monitoring of the structural and cellular organisation of developing embryos and the subcellular localisation of the Esr proteins by immunofluorescence and immunogold labelling. The results obtained using confocal and electron microscopy revealed that Esr proteins were localised in elements of the secretory pathway and cell walls in microspore-derived embryo cells during early embryogenesis. Esr proteins were also detected in the liquid medium of maize microspore cultures and accumulated at 20 days of culture. Tunicamycin treatment to block protein glycosilation and, therefore, secretion inhibited microspore-derived embryo development, which was subsequently recovered by supplementation with medium containing all the secreted factors from a well developed microspore culture. Esr labelling was not present in non-developing microspore embryos of cultures treated with tunicamycin, whereas labelling was present again in the Golgi elements and secretory vesicles of embryo cells when development was restored. The results indicate that Esr proteins are part of the secreted proteins, which show a nursing or signalling role during in vitro embryo development in maize microspore embryogenesis cultures and provide new evidence for an endosperm-like function of microspore-derived embryo structures during the early stages.

Abstract Background In inflammatory bowel diseases (IBD), epithelial barrier defects occur as a consequence of chronic inflammation. Recent research suggested that cell polarity alterations may be upstream of barrier defects and additionally play a role in IBD-associated carcinogenesis (colitis-associated and small intestinal carcinoma). Par4 is a gene encoding a protein crucial in the development of cell polarity. LKB1, its human homologue, is mutated in Peutz–Jeghers syndrome (PJS), a genetic condition characterised by a higher risk of epithelial cancers. While the pivotal role of Par4/LKB1 in the development of epithelial cell polarity is established, its involvement in IBD-associated barrier defects and carcinogenesis is yet to be defined. Methods Endoscopic bowel mucosa samples from patients with Crohn’s disease, ulcerative colitis, PJS and controls were analysed to assess expression and localisation of Par4/LKB1. Cryosections were immunostained for Par4/LKB-1 along with a diverse set of markers of cell polarity and intercellular adhesion. The analysis was performed by confocal laser scanning microscopy in order to compare the expression of mucosal as well as the subcellular localisation of Par4/LKB1 in the gut mucosa. A quantitative analysis of protein expression with western blotting was performed as well. The function of Par4/LKB1 in intestinal epithelial cells (IEC) was evaluated by means of a Par4-deficient IEC model as well as in an IEC Par4-overexpression model. Moreover, a preliminary assessment of the possible role of Par4/LKB1-related polarity processes in IBD-associated carcinomas was performed through scanning genetic data from colitis-associated carcinomas for potential Par4-related mutations and altered Par4/LKB1 expression. Results The immunofluorescent staining allowed visualisation of intracellular expression of Par4 in epithelia from PJS, IBD patients and controls. In PJS-polyps, despite the alteration of regular tissue architecture typical of these lesions, the polarity of epithelial cells was maintained - contrary to control tissue, a punctate pattern of the Par4 staining was shown. In IBD tissue, no relevant differences in Par4 expression at confocal microscopy, as well as a quantitative assessment, were observed as compared with controls. No differences were observed in PJS or IBD samples as regards the expression of cell polarity markers such as Par3, CD71, crb3 or adhesion molecules such as ZO-1, e-cadherin, occludin, JAM-A. As regards the IEC model, we observed that Caco-2/BBe cells overexpressing Par4/LKB1 showed enhanced Par4/LKB1 cytosolic staining at immunofluorescence. In this model, a defective epithelial polarity and organisation on permeable supports could be observed in Caco2/BBe cysts in parallel with reduced expression of native LKB1 as seen in western blots. Preliminary analysis of data from our colitis-associated carcinomas database suggested various pathways potentially involving Par4/LKB1, yet no direct analysis of tissue samples for Par4-related mutations was performed yet. Conclusions Altered expression of Par4 in epithelial cells may influence the development of a functional epithelial barrier as suggested by IEC models. Par4/LKB1 as a known oncosuppressor gene is involved in a number of pathways potentially relevant for carcinogenesis in IBD; however, a direct connection between Par4-related alteration of the epithelial barrier and carcinogenesis is yet to be established.

One of the last Nobel Prizes for Medicine and Physiology of this century was awarded to Gunther Blobel for his pioneering work on the mechanisms of secretion in eukaryotic and prokaryotic cells. This prize for molecular cell biology followed that awarded to George Palade, Christian DeDuve and Albert Claude in 1974 and demonstrated that the control of the subcellular localization of proteins is achieved by molecular machines that read ‘addresses’ that are embedded in the primary sequence of a protein. In the case of secretory proteins, these addresses are known as signal sequences. Sorting of proteins from their site of synthesis to their final destination is now known to be a key cellular process, which, if impaired, can give rise to lethal defects. Gunther Blobel and his group established an experimental approach that is now used by numerous researchers to study secretory pathways. The analysis of the secretory pathway by George Palade and his collaborators in the 1960s took advantage of advanced ultrastructural analysis and cell-fractionation procedures. Subsequently, in the 1970s, a number of investigators recognized that further studies at the molecular level were required. For this purpose, Gunther Blobel, together with Bernhard Dobberstein, set up an assay that was one of the first to reconstitute in vitro the translocation of a nascent polypeptide chain across vesicles derived from the rough endoplasmic reticulum. This in vitro assay led the researchers to establish the signal-peptide hypothesis, which was first proposed by David Sabatini and Gunther Blobel in 1971. The complete characterization of the translocation machinery itself, which is composed of ribonucleoprotein complexes and integral membrane proteins, was achieved after much effort and imaginative studies performed by several research groups from different countries. The in vitro assay not only was used by numerous investigators in the field but it also provided the conceptual framework necessary for unravelling of the molecular machinery involved in the different steps of the intracellular transport of proteins. At the beginning of the 1980s, James Rothman and his colleagues developed another type of in vitro assay, which was based upon the use of fractions derived from different membrane compartments and cytosolic extracts. The goal of these researchers was to reconstitute the specific fusion between a donor membrane and an acceptor membrane. In less than one decade, the assay was refined to the point where it became a very powerful tool for biochemical characterization of cellular machines that move proteins from one organelle to another. Membrane-fusion assays allow the identification not only of protein complexes but also of the energy requirements of the fusion events. Several groups who focus on the endocytic pathway have subsequently developed similar assays. While the biochemical experiments were in progress, Randy Scheckman and his colleagues launched a genetic approach to study secretory pathways, using yeast as a model system. Because of the high degree of conservation of this essential cellular function, gene product(s) identified in yeast have mammalian homologues that were characterised simultaneously by the biochemical approaches described above. Together, these strategies gave rise to a molecular description of the minimal protein machinery necessary for the numerous transport pathways in eukaryotic cells. As with all experimental methods designed by biologists, however, even the powerful reconstitution and genetic approaches had their limitations. Fortunately, another original strategy emerged, in which cells could be permeabilized by physical trauma or chemical treatments so that small holes appeared in the plasma membrane. This procedure empties cells of their cytoplasmic components but maintains their structural organization and allows investigators to reintroduce well-defined products such as activator(s) or inhibitor(s). The permeabilized-cell strategy has opened new possibilities for future studies, because one now has biochemical access to information contained in the structural organisation of cells - information that is lost in cell-extract and genetics-based studies. Most investigators in the field had faith in the specificity of the biochemical reactions of the transport processes and were less interested in the efficiency of the process in vivo. The importance of this lapse of attention to detail became evident when the reaction rates in the in vitro systems were compared with those during normal physiological membrane transport in vivo. Consequently, it was only after the discovery of molecular motors that reaction rates were studied seriously and the role of motor proteins was considered in experiments designed by groups interested in molecular analysis of membrane trafficking. However, one should note the interest of those who developed in vitro assays for membrane transport in studies of the roles of molecular motors in movement of intracellular cargo along microtubules or microfilaments. The myriad of molecular motors that move the various molecular cargos along cellular tracks in opposite directions is bewildering. Another challenge for the future, well illustrated by studies that take advantage of recombinant DNA technology, will be to describe the spatial and temporal plasticity of the protein that make up machines that participate in vesicular trafficking. In this respect, the use of chimeric proteins tagged with the green fluorescent protein(s) is already very significant but still only in its infancy. The systematic use of videomicroscopy recording, which allows subsequent quantitative and multiparametric analysis at the single-cell or multicellular level, has allowed development of new perspectives on membrane trafficking. Our knowledge of the numerous proteins capable of forming molecular machines will be facilitated by the complete description of the genomes of model organisms - whether or not we study them in a unicellular or a multicellular context. Looking ahead, more research should be done on membrane trafficking and its regulation by the cytoskeleton in different cell types. Although numerous studies have already been performed on polarized cells, such as epithelial cells and neurons, many differentiated cell types have not been investigated at all. Moreover studies of cell differentiation during development or during physiopathological processes are still scarce and, here too, more research is needed to characterise the molecular features of trafficking during these processes. The pioneering work by numerous investigators over the past thirty years indeed provides a solid basis for future investigation in this research area. Certainly, these illuminating studies have opened new avenues and point to exciting challenges for the next generation of molecular cell biologists, who will experience even more spectacular discoveries.

AbstractThe endoplasmic reticulum (ER) is a eukaryotic subcellular organelle composed of tubules and sheet-like areas of membrane connected at junctions. The tubule network is highly dynamic and undergoes rapid and continual rearrangement. There are currently few tools to evaluate network organisation and dynamics. We quantified ER network organisation in Vero and MRC5 cells, and developed an analysis workflow for dynamics of established tubules in live cells. The persistence length, tubule length, junction coordination number and angles of the network were quantified. Hallmarks of imbalances in ER tension, indications of interactions with microtubules and other subcellular organelles, and active dynamics were observed. Clear differences in dynamic behaviour were observed for established tubules at different positions within the cell using itemset mining. We found that tubules with activity-driven fluctuations were more likely to be located away from the cell periphery and a population of peripheral tubules with no signs of active motion was found.