Deconstructing heterogeneity of replicative senescence in human mesenchymal stem cells at single cell resolution.

Following prolonged cell division, mesenchymal stem cells enter replicative senescence, a state of permanent cell cycle arrest that constrains the use of this cell type in regenerative medicine applications and that in vivo substantially contributes to organismal ageing. Multiple cellular processes such as telomere dysfunction, DNA damage and oncogene activation are implicated in promoting replicative senescence, but whether mesenchymal stem cells enter different pre-senescent and senescent states has remained unclear. To address this knowledge gap, we subjected serially passaged human ESC-derived mesenchymal stem cells (esMSCs) to single cell profiling and single cell RNA-sequencing during their progressive entry into replicative senescence. We found that esMSC transitioned through newly identified pre-senescent cell states before entering into three different senescent cell states. By deconstructing this heterogeneity and temporally ordering these pre-senescent and senescent esMSC subpopulations into developmental trajectories, we identified markers and predicted drivers of these cell states. Regulatory networks that capture connections between genes at each timepoint demonstrated a loss of connectivity, and specific genes altered their gene expression distributions as cells entered senescence. Collectively, this data reconciles previous observations that identified different senescence programs within an individual cell type and should enable the design of novel senotherapeutic regimes that can overcome in vitro MSC expansion constraints or that can perhaps slow organismal ageing.

Observational study investigating Tolerance Of Anticancer Systemic Therapy In the Elderly (TOASTIE): a protocol.

INTRODUCTION: The number of older adults diagnosed with cancer is increasing. Older adults are more likely to have pre-existing frailty, which is associated with greater chemotherapy-related toxicity. Early identification of those at risk of toxicity is important to reduce patient morbidity and mortality. Current chemotherapy toxicity prediction tools including the Cancer and Ageing Research Group (CARG) tool exist but are not in routine clinical use and have not been prospectively validated in a UK population. This study is the first prospective study to investigate the CARG tool in a UK population with cancer. METHODS AND ANALYSIS: Tolerance Of Anticancer Systemic Therapy In the Elderly is a prospective observational study of patients, aged ≥65 years, commencing first-line (any indication) chemotherapy for a solid-organ malignancy. Patients receiving other systemic anticancer agents or radiotherapy will be excluded. The primary objective will be to validate the ability of the CARG score to predict grade 3+ toxicity in this population. Secondary objectives include describing the feasibility of screening for frailty, as well as the prevalance of frailty in this population and assessing patient and clinician perception of chemotherapy toxicity risk. 500 patients will be recruited over a two year period. Baseline assessments will be recorded. At the end of the 6-month follow-up period, toxicity data will be retrospectively collected. A descriptive analysis of the recruited population will be performed. The validity of the CARG model will be analysed using receiver-operating characteristic curves and calculation of the area under the curve (c-statistic). ETHICS AND DISSEMINATION: The study has received ethical approval from the East of Scotland Research Ethics Service 20/ES/0114. Results will be reported in peer-reviewed scientific journals and disseminated to patient organisations and media.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

The impact of wrist fracture, surgical repair and immobilization on patients: a qualitative study.

OBJECTIVE: To investigate patients' experience following wrist fracture, surgical repair and immobilization. DESIGN: A qualitative investigation involving individual participant interviews. SETTING: A metropolitan trauma service. SUBJECTS: In all, 31 participants were consecutively recruited from three groups within a randomized controlled trial comparing immobilization for one ( n = 11), three ( n = 10) or six weeks ( n = 10) following surgical treatment for wrist fracture. INTERVENTION: Individual interviews were conducted within three months of cast removal. Questions prompted discussion of the experience of fracture, surgery and immobilization. Interviews were audio-recorded, transcribed verbatim. At least two independent researchers performed coding and theming following principles of thematic analysis. RESULTS: Two themes were identified: (1) impact of the injury varies widely and (2) health care consumers want trustworthy dialogue. Participant reports indicated that recovery from wrist fracture, surgery and immobilization is challenging with significant changes to social role and increased dependence. For many, lack of empathy from health professionals and limited acknowledgement of the personal impact of injury led to dissatisfaction. Health professionals did not consistently tailor communication or adopt strategies to address specific needs for pain management, education and support requirements. There was no evidence that processes were implemented to enhance participant recall and comprehension. Most participants experienced their cast as a barrier to function. However, within the group of participants immobilized for one week, a number felt the cast was removed too soon. CONCLUSION: Participant reports indicate that recovery from surgically repaired wrist fracture is challenging. Opportunities exist to refine care in pain management, education and active engagement of patients in their care.

HIF-2α Promotes Dissemination of Plasma Cells in Multiple Myeloma by Regulating CXCL12/CXCR4 and CCR1.

Disease progression and relapse in multiple myeloma is dependent on the ability of the multiple myeloma plasma cells (PC) to reenter the circulation and disseminate throughout the bone marrow. Increased bone marrow hypoxia is associated with increased recirculation of multiple myeloma PCs. Accordingly, we hypothesized that during chronic hypoxia, activation of HIF-2α may overcome the bone marrow retention signal provided by stromal-derived CXCL12, thereby enabling dissemination of multiple myeloma PCs. Here we demonstrate that HIF-2α upregulates multiple myeloma PC CXCL12 expression, decreasing migration toward CXCL12 and reducing adhesion to mesenchymal stromal cells in vitro We also found that HIF-2α strongly induced expression of the chemokine receptor CCR1 in multiple myeloma PCs. CCR1 activation potently induces multiple myeloma PC migration toward CCL3 while abrogating the multiple myeloma PC migratory response to CXCL12. In addition, increased CCR1 expression by multiple myeloma PCs conferred poor prognosis in newly diagnosed multiple myeloma patients and was associated with an increase in circulating multiple myeloma PCs in these patients. Taken together, our results suggest a role for hypoxia-mediated CCR1 upregulation in driving the egress of multiple myeloma PCs from the bone marrow. Targeting CCR1 may represent a novel strategy to prevent dissemination and overt relapse in multiple myeloma. Cancer Res; 77(20); 5452-63. ©2017 AACR.

ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network.

In neurosecretory cells, myosin VI associated with secretory granules (SGs) mediates their activity-dependent recruitment to the cortical actin network and is necessary to sustain exocytosis. The mechanism by which myosin VI interacts with SGs is unknown. Using a myosin VI pull-down assay and mass spectrometry we identified Mena, a member of the ENA/VASP family, as a myosin VI binding partner in PC12 cells, and confirmed that Mena colocalized with myosin VI on SGs. Using a knock-sideways approach to inactivate the ENA/VASP family members by mitochondrial relocation, we revealed a concomitant redistribution of myosin VI. This was ensued by a reduction in the association of myosin VI with SGs, a decreased SG mobility and density in proximity to the plasma membrane as well as decreased evoked exocytosis. These data demonstrate that ENA/VASP proteins regulate SG exocytosis through modulating the activity of myosin VI.

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones.

Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs.

Tetraspanin 7 (TSPAN7) expression is upregulated in multiple myeloma patients and inhibits myeloma tumour development in vivo.

BACKGROUND: Increased expression of the tetraspanin TSPAN7 has been observed in a number of cancers; however, it is unclear how TSPAN7 plays a role in cancer progression. METHODS: We investigated the expression of TSPAN7 in the haematological malignancy multiple myleoma (MM) and assessed the consequences of TSPAN7 expression in the adhesion, migration and growth of MM plasma cells (PC) in vitro and in bone marrow (BM) homing and tumour growth in vivo. Finally, we characterised the association of TSPAN7 with cell surface partner molecules in vitro. RESULTS: TSPAN7 was found to be highly expressed at the RNA and protein level in CD138(+) MM PC from approximately 50% of MM patients. TSPAN7 overexpression in the murine myeloma cell line 5TGM1 significantly reduced tumour burden in 5TGM1/KaLwRij mice 4 weeks after intravenous adminstration of 5TGM1 cells. While TSPAN7 overexpression did not affect cell proliferation in vitro, TSPAN7 increased 5TGM1 cell adhesion to BM stromal cells and transendothelial migration. In addition, TSPAN7 was found to associate with the molecular chaperone calnexin on the cell surface. CONCLUSION: These results suggest that elevated TSPAN7 may be associated with better outcomes for up to 50% of MM patients.

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking.

In neurosecretory cells, secretory vesicles (SVs) undergo Ca(2+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking.

An acto-myosin II constricting ring initiates the fission of activity-dependent bulk endosomes in neurosecretory cells.

Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact-GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells.

The effect of the PI3K inhibitor BKM120 on tumour growth and osteolytic bone disease in multiple myeloma.

The plasma cell malignancy multiple myeloma (MM) is unique amongst haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts. NVP-BKM120 (Buparlisib, Novartis) is a PI3K inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we have examined the anti-tumorigenic effects of BKM120 in an immunocompetent mouse model of MM and its effects on osteoblast and osteoclast formation and function. BKM120 treatment (40 mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and µCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BKM120-treated animals. BKM120 also mediated a significant increase in serum levels of the osteoblast marker P1NP, and a significant decrease in serum levels of the osteoclast marker TRAcP5. In vitro, BKM120 decreased MM plasma cell proliferation, osteoclast formation and function, and promoted osteoblast formation and function. These findings suggest that, in addition to its anti-tumour properties, BKM120 could be used to treat osteolytic bone disease in MM patients.

The effect of the dual PI3K and mTOR inhibitor BEZ235 on tumour growth and osteolytic bone disease in multiple myeloma.

The plasma cell malignancy multiple myeloma (MM) is unique among haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt/mTOR pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts. NVP-BEZ235 is a dual pan class I PI3K and mTOR inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we examined the anti-tumorigenic effects of BEZ235 in an immunocompetent mouse model of MM and assessed the effects of BEZ235 on osteoblast and osteoclast formation and function. BEZ235 treatment (50 mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and µCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BEZ235-treated animals. Levels of the serum osteoblast marker P1NP were significantly higher in BEZ235-treated animals, while levels of the osteoclast marker TRAcP5 were reduced. In vitro, BEZ235 decreased MM plasma cell proliferation, osteoclast formation and function and promoted osteoblast formation and function. These findings suggest that, in addition to its anti-tumour properties, BEZ235 could be useful in treating osteolytic bone disease in MM patients.

Brief report: the differential roles of mTORC1 and mTORC2 in mesenchymal stem cell differentiation.

Adipocytes (AdCs) and osteoblasts (OBs) are derived from mesenchymal stem cells (MSCs) and differentiation toward either lineage is both mutually exclusive and transcriptionally controlled. Recent studies implicate the mammalian target of rapamycin (mTOR) pathway as important in determining MSC fate, with inhibition of mTOR promoting OB differentiation and suppressing AdC differentiation. mTOR functions within two distinct multiprotein complexes, mTORC1 and mTORC2, each of which contains the unique adaptor protein, raptor or rictor, respectively. While compounds used to study mTOR signaling, such as rapamycin and related analogs, primarily inhibit mTORC1, prolonged exposure can also disrupt mTORC2 function, confounding interpretation of inhibitor studies. As a result, the relative contribution of mTORC1 and mTORC2 to MSC fate determination remains unclear. In this study, we generated primary mouse MSCs deficient in either Rptor (RapKO) or Rictor (RicKO) using the Cre/loxP system. Cre-mediated deletion of Rptor or Rictor resulted in impaired mTORC1 and mTORC2 signaling, respectively. Under lineage-inductive culture conditions, RapKO MSCs displayed a reduced capacity to form lipid-laden AdCs and an increased capacity to form a mineralized matrix. In contrast, RicKO MSCs displayed reduced osteogenic differentiation capacity and enhanced adipogenic differentiation potential. Taken together, our findings reveal distinct roles for mTORC1 and mTORC2 in MSC lineage commitment.

Caveolin-1 is necessary for hepatic oxidative lipid metabolism: evidence for crosstalk between caveolin-1 and bile acid signaling.

Caveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1-/- mice exhibited impaired hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1-/- mice involve impaired PPARα ligand signaling and attenuated bile acid and FXRα signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPARα, FXRα, and SHP) and bile acid signaling.

The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane.

Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1(Δ317-333)) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1(Δ317-333) to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1(Δ317-333) binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1(Δ317-333) could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.

Inhibition of PIKfyve by YM-201636 dysregulates autophagy and leads to apoptosis-independent neuronal cell death.

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P 2, results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death.

Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.

Caveolae, lipid droplets, and adipose tissue biology: pathophysiological aspects.

Adipocytes are specialized cells that function to store energy in the form of lipids, predominantly triglycerides (TGs), and as a regulatory system contributing to metabolic homoeostasis through the production and secretion of hormones and cytokines. The regulation of lipid homeostasis by adipose tissue is an important aspect of whole-body metabolism. Owing to the central nature of adipose tissue in lipid metabolism, dysregulation has wide-ranging effects, contributing to disorders as diverse as diabetes, cardiovascular disease, cancer, and neurodegeneration. Excess lipids are stored in specialized organelles called lipid droplets (LDs). The surface of the lipid droplet can be considered a highly regulated membrane domain that both protects the contents of the LD from unregulated lipolysis and the cell from the cytotoxic effects of elevated free fatty acids. The surface of the LD is coated with a variety of regulatory proteins, either resident or transiently associated, including enzymes involved in the breakdown of TG, lipid transport proteins, and cofactors. Recent studies have begun to unravel the range of LD-associated proteins and to define their functional significance. Importantly, the involvement of LD proteins in pathophysiological disorders is beginning to be understood. This review will outline recent advances in defining the diversity of LD-associated proteins and their links to metabolic disorders including the integral membrane protein, caveolin-1 (CAV1). Analysis of the role of CAV1 in adipose tissue has highlighted the interconnectedness between the regulation of lipid storage and the function of the adipocyte plasma membrane.

Caveolin-1 deficiency leads to increased susceptibility to cell death and fibrosis in white adipose tissue: characterization of a lipodystrophic model.

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1(-/-) mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1(-/-) adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1(+/+) mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1(-/-) mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.

Postlipolytic insulin-dependent remodeling of micro lipid droplets in adipocytes.

Despite the lipolysis-lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology. We characterized mLD formation in cultured adipocytes, and in primary adipocytes isolated from mouse epididymal fat pads, in response to acute activation of lipolysis. Using real-time quantitative imaging and electron tomography, we show that formation of mLDs in cultured adipocytes occurs throughout the cell to increase total LD surface area by ~30% but does not involve detectable fission from large LDs. Peripheral mLDs are monolayered structures with a neutral lipid core and are sites of active lipolysis. Electron tomography reveals preferential association of mLDs with the endoplasmic reticulum. Treatment with insulin and fatty acids results in the reformation of macroLDs and return to the basal state. Insulin-dependent reformation of large LDs involves two distinct processes: microtubule-dependent homotypic fusion of mLDs and expansion of individual mLDs. We identify a physiologically important role for LD fusion that is involved in a reversible lipolytic cycle in adipocytes.