ATR mediates a checkpoint at the nuclear envelope in response to mechanical stress.

ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes.

LncRNA EPR regulates intestinal mucus production and protects against inflammation and tumorigenesis.

The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes.

The E-Syt3 cleavage and traffic uncovers the primordial cisterna, a new organelle that mothers the lipid droplets in the adipocyte.

Extended synaptotagmins are endoplasmic reticulum proteins consisting of an SMP domain and multiple C2 domains that bind phospholipids and Ca2+ . E-Syts create contact junctions between the ER and plasma membrane (PM) to facilitate the exchange of glycerophospholipids between the apposed membranes. We find in the differentiating adipocyte that the E-Syt3 carboxyl domain is cleaved by a multi-step mechanism that includes removing the C2C domain. Confocal and live-cell time-lapse studies show that truncated E-Syt3ΔC2C, as well as endogenous E-Syt3 and the coat protein PLIN1, target the LDs from an annular, single giant ER cisterna. Inhibition of the proteasome blocks the proteolytic cleavage of Esyt3 and E-Syt3ΔC2C and causes the E-Syt3ΔC2C retention in the giant cisterna. The Esyt3 and PLIN1 distributions and LDs biogenesis show that the primordial cisterna, as we call it, is the birth and nurturing site of LDs in the adipocyte. Isoproterenol-induced lipolysis results in loss of cytoplasmic LDs and reappearance of the primordial cisterna. Electron microscopy and 3D-electron tomography studies show that the primordial cisterna consists of a tightly packed network of varicose tubules with extensively blistered membranes. Rounds of homotypic fusions from nascent to mature LDs play a central role in LD growth. The knockdown of E-Syt3 inhibits LD biogenesis. The identification of the primordial cisterna, an organelle that substitutes the randomly scattered ER foci that mother the LDs in non-adipose cells, sets the stage for a better understanding of LD biogenesis in the adipocyte.

Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer.

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1.

The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral-facial-digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein Cep164. Myosin VI depletion in non-tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be, at least partially, ascribed to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.

GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis.

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.

Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/ß-catenin signaling.

Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/ß-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/ß-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/ß-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/ß-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/ß-catenin activity.

The Regulated Secretion and Models of Intracellular Transport: The Goblet Cell as an Example.

Transport models are extremely important to map thousands of proteins and their interactions inside a cell. The transport pathways of luminal and at least initially soluble secretory proteins synthesized in the endoplasmic reticulum can be divided into two groups: the so-called constitutive secretory pathway and regulated secretion (RS) pathway, in which the RS proteins pass through the Golgi complex and are accumulated into storage/secretion granules (SGs). Their contents are released when stimuli trigger the fusion of SGs with the plasma membrane (PM). In specialized exocrine, endocrine, and nerve cells, the RS proteins pass through the baso-lateral plasmalemma. In polarized cells, the RS proteins secrete through the apical PM. This exocytosis of the RS proteins increases in response to external stimuli. Here, we analyze RS in goblet cells to try to understand the transport model that can be used for the explanation of the literature data related to the intracellular transport of their mucins.

Intracellular Membrane Transport in Vascular Endothelial Cells.

The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.

COVID-19 Biogenesis and Intracellular Transport.

SARS-CoV-2 is responsible for the COVID-19 pandemic. The structure of SARS-CoV-2 and most of its proteins of have been deciphered. SARS-CoV-2 enters cells through the endocytic pathway and perforates the endosomes' membranes, and its (+) RNA appears in the cytosol. Then, SARS-CoV-2 starts to use the protein machines of host cells and their membranes for its biogenesis. SARS-CoV-2 generates a replication organelle in the reticulo-vesicular network of the zippered endoplasmic reticulum and double membrane vesicles. Then, viral proteins start to oligomerize and are subjected to budding within the ER exit sites, and its virions are passed through the Golgi complex, where the proteins are subjected to glycosylation and appear in post-Golgi carriers. After their fusion with the plasma membrane, glycosylated virions are secreted into the lumen of airways or (seemingly rarely) into the space between epithelial cells. This review focuses on the biology of SARS-CoV-2's interactions with cells and its transport within cells. Our analysis revealed a significant number of unclear points related to intracellular transport in SARS-CoV-2-infected cells.

The Diffusion Model of Intra-Golgi Transport Has Limited Power.

The Golgi complex (GC) is the main station along the cell biosecretory pathway. Until now, mechanisms of intra-Golgi transport (IGT) have remained unclear. Herein, we confirm that the goodness-of-fit of the regression lines describing the exit of a cargo from the Golgi zone (GZ) corresponds to an exponential decay. When the GC was empty before the re-initiation of the intra-Golgi transport, this parameter of the curves describing the kinetics of different cargoes (which are deleted in Golgi vesicles) with different diffusional mobilities within the GZ as well as their exit from the GZ was maximal for the piecewise nonlinear regression, wherein the first segment was horizontal, while the second segment was similar to the exponential decay. The kinetic curve describing cargo exit from the GC per se resembled a linear decay. The Monte-Carlo simulation revealed that such curves reflect the role of microtubule growth in cells with a central GC or the random hovering of ministacks in cells lacking a microtubule. The synchronization of cargo exit from the GC already filled with a cargo using the wave synchronization protocol did not reveal the equilibration of cargo within a Golgi stack, which would be expected from the diffusion model (DM) of IGT. Moreover, not all cisternae are connected to each other in mini-stacks that are transporting membrane proteins. Finally, the kinetics of post-Golgi carriers and the important role of SNAREs for IGT at different level of IGT also argue against the DM of IGT.

Activation of Src family kinase ameliorates secretory trafficking in mutant prion protein cells.

Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.

PillarX: A Microfluidic Device to Profile Circulating Tumor Cell Clusters Based on Geometry, Deformability, and Epithelial State.

Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.

JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function.

RATIONALE: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. OBJECTIVE: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. METHODS AND RESULTS: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of ß-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. CONCLUSIONS: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

Opinion: On the Way towards the New Paradigm of Atherosclerosis.

Atherosclerosis is a multicausal disease characterized by the formation of cholesterol-containing plaque in the pronounced intima nearest to the heart's elastic-type arteries that have high levels of blood circulation. Plaques are formed due to arterial pressure-induced damage to the endothelium in areas of turbulent blood flow. It is found in the majority of the Western population, including young people. This denies the monogenic mechanism of atherogenesis. In 1988, Orekhov et al. and Kawai et al. discovered that the presence of atherogenic (modified, including oxidized ones) LDLs is necessary for atherogenesis. On the basis of our discovery, suggesting that the overloading of enterocytes with lipids could lead to the formation of modified LDLs, we proposed a new hypothesis explaining the main factors of atherogenesis. Indeed, when endothelial cells are damaged and then pass through the G2 phase of their cell cycle they secrete proteins into their basement membrane. This leads to thickening of the basement membrane and increases its affinity to LDL especially for modified ones. When the enterocyte transcytosis pathway is overloaded with fat, very large chylomicrons are formed, which have few sialic acids, circulate in the blood for a long time, undergo oxidation, and can induce the production of autoantibodies. It is the sialic acids that shield the short forks of the polysaccharide chains to which autoantibodies are produced. Here, these data are evaluated from the point of view of our new model.

Comparison of the Cisterna Maturation-Progression Model with the Kiss-and-Run Model of Intra-Golgi Transport: Role of Cisternal Pores and Cargo Domains.

The Golgi complex is the central station of the secretory pathway. Knowledge about the mechanisms of intra-Golgi transport is inconsistent. Here, we compared the explanatory power of the cisterna maturation-progression model and the kiss-and-run model. During intra-Golgi transport, conventional cargoes undergo concentration and form cisternal distensions or distinct membrane domains that contain only one membrane cargo. These domains and distension are separated from the rest of the Golgi cisternae by rows of pores. After the arrival of any membrane cargo or a large cargo aggregate at the Golgi complex, the cis-Golgi SNAREs become enriched within the membrane of cargo-containing domains and then replaced by the trans-Golgi SNAREs. During the passage of these domains, the number of cisternal pores decreases. Restoration of the cisternal pores is COPI-dependent. Our observations are more in line with the kiss-and-run model.

Propranolol Reduces the Development of Lesions and Rescues Barrier Function in Cerebral Cavernous Malformations: A Preclinical Study.

[Figure: see text].

Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier.

RATIONALE: The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/ß-catenin signaling is responsible for the early phases of brain vascularization and BBB differentiation. However, this signal declines after birth, and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown. OBJECTIVE: Sox17 (SRY [sex-determining region Y]-box 17) constitutes a major downstream target of Wnt/ß-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/ß-catenin in inducing BBB differentiation and maintenance. METHODS AND RESULTS: Using reporter mice and nuclear staining of Sox17 and ß-catenin, we report that although ß-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo and progressively declines after birth. In search of Sox17 mechanism of action, RNA sequencing analysis of gene expression of brain endothelial cells has identified members of the Wnt/ß-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/ß-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the ß-catenin destruction complex or expression of a degradation-resistant ß-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17. CONCLUSIONS: Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB, and they underline the strict reciprocal tuning of this transcription factor and Wnt/ß-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.

An essential step of kinetochore formation controlled by the SNARE protein Snap29.

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. Here, we identify a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis-segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. We propose that part of the outer kinetochore is functionally similar to membrane fusion interfaces.

Structure of the enterocyte transcytosis compartments during lipid absorption.

In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view.