Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.

The present study was undertaken to investigate the effects of the T-type calcium channel blocker, Mibefradil, on white adipocytes. Unexpected for a T-type channel blocker, Mibefradil was found to increase intracellular calcium levels, cause lipid droplet fusion, and result in cell death. Calcium imaging of white adipocytes showed an increase of calcium concentration by Mibefradil at concentrations ranging from10-50 µM. The elevation in calcium by Mibefradil was significantly reduced by pretreatment of cells with Thapsigargin, an endoplasmic reticulum (ER) specific Ca ATPase inhibitor. Additionally, lipid droplet fusion and cell death were also attenuated by Thapsigargin pretreatment in white adipocytes. We conclude that Mibefradil elevated intracellular calcium levels, induced lipid droplet fusion and cell death in white adipocytes via mobilizing intracellular calcium stores from the ER. These results describe novel effects of Mibefradil on white adipocytes and may provide new insight into how this drug might be repurposed in obesity research.

Mitochondria play a central role in lipid metabolism and pathology in obesity and type 2 diabetes mellitus. Mitochondria have been shown to associate with lipid droplets (LDs) in multiple tissues but the functional role of these peridroplet mitochondria (PDM) is unknown. This work reveals that PDM have unique protein composition and cristae structure, and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities. (2) PDM have reduced beta oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and beta oxidation. (3) PDM support LD expansion as Perilipin 5-induced recruitment of mitochondria to LDs increases ATP-dependent triacylglyceride synthesis. (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis. We suggest that increased mitochondrial recruitment to LDs may be part of a generalized adaptive response in physiological conditions that require LD expansion, such as post-prandial lipid synthesis and storage. Furthermore, PDM-mediated LD expansion may play a role in muscle and liver injury from lipotoxicity in conditions of nutrient excess, such as obesity and hyperlipidemia. A better understanding of PDM and LD biology may therefore lead to new therapies for lipotoxic tissue injury and insulin resistance. / 2020-10-24T00:00:00Z

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Molecular motor proteins are responsible for the long range transport of vesicles and organelles inside living cells. A small number of motor types transport thousands of distinct cargoes to various regions in the cell at the same time. This requires that intracellular transport be tightly regulated, yet the details of how motor regulators and cofactors tune motor function remain unknown in most cases. In-vitro studies at the single motor level have been instrumental in understanding the function of individual motors. In this thesis work I developed the methodology to extend in-vitro experiments to interrogate motor regulation at the single molecule level. I describe my modifications to the microscope setup as well as the acquisition cycle that made this possible. By combining differential interference contrast microscopy with single molecule fluorescence imaging and optical trapping I was able to manipulate and image the cargo while imaging a fluorescently-labeled regulator binding at the site of the motors. I used lipid droplets purified from Drosophila embryos as cargoes. Lipid droplets are carried by the opposite polarity microtubule motors kinesin and dynein in the embryos, and bind specifically to microtubules in-vitro. In the presence of ATP they exhibit long-range and short-range motility. For this proof-of-principle experiment I used fluorescently labeled AMPPNP, a non-hydrolysable analogue of ATP which binds to the motor domain of kinesin when microtubule-bound, to image the binding of the nucleotide to the motor and demonstrate the activity of the motors. While a large fraction of microtubule-bound droplets co-localized with a fluorescent AMPPNP molecule, non-specific binding of the nucleotide to the microscope slide surface prevented confirming the specificity of the colocalization events. Nevertheless, these data demonstrate the ability of the methodology to capture, in real time, the process of a regulator binding the motor at the single molecule level. / text

The protein Hedgehog (Hh) is a highly conserved, secreted ligand (and morphogen) capable of patterning many different tissues during development. Recently, Sonic Hedgehog (SHH) a human homolog of Drosophila Hh was found to be a causative agent in certain cancers. While several drugs are being developed to combat the binding of SHH to its receptor Patched or the Patched-target Smoothened, very little is known about how SHH is secreted from the producing cell, another site for therapeutic targeting. We report here the characterization of a novel proteolytic event and genetic pathway that controls Hh intracellular sorting and axon transport using the Drosophila eye imaginal disc as our model system. In fly larval photoreceptor neurons the developmental signal Hh is guided to the apical (retina) and basal (growth cone, GC) ends where secretion of the morphogen is an inductive factor in photoreceptor differentiation and establishment of eye/brain neural connections. The Hh secreted from the basal side induces lamina development while Hh secreted at the retina induces ommatidial development. Hedgehog processing consists of autocleavage from its 46 kDa form (HhU) to become a lipid-modified N-terminal signaling molecule (HhN; 19kDa) and a C-terminal molecule (HhC24; 24 kDa). Following autocleavage, a fraction of the C-terminal auto-cleavage product then undergoes a second cleavage event leading to 16 kDa (HhC16) and 9 kDa products. Nothing is known about the significance of the C-terminal “2nd cleavage” other than its occurrence in both fly and human tissue. In an effort to identify regulators of Hh sorting, we discovered that the HhC “2nd cleavage” is a determining factor in the sorting of the HhN signaling domain. That is, if a cell induces more cleavage (more HhC16) we observe more HhN in the apical domain. Likewise, if a cell inhibits 2nd cleavage (less HhC16) we see more basal HhN. Creation of a “2nd cleavage mutant” shows that this process has developmental significance. Further, biochemical characterization of the 2nd cleavage suggests it occurs in the ER after autocleavage and that HhC24 can exit the cell in a Golgi independent manner (via lipid droplets) while HhC16 remains intracellular. The ER exit of HhC24 appears to be controlled by a conserved PP2A (Mts) /PKB (Akt) kinase pathway which potentially regulates the size and number of lipid droplets produced. These findings are an important first step in understanding the intracellular sorting and transport of Hh and highlight new targets for the treatment of SHH-related cancers. The discovery of divergent modes of Hh secretion and the “2nd cleavage” open novel avenues for Hh research by offering an alternative, and very direct, line of attack in the treatment of Hh-related cancer.

No description available.

The putative peroxisome proliferator-activated receptor (PPAR) α ligand, conjugated linoleic acid (CLA) induced cytoplasmic lipid droplet (LD) formation in H4IIE rat hepatoma cells. Currently, the mechanism(s) by which CLA isomers affects hepatic LD formation is unclear. We have investigated the role of PPARα and fatty acid (FA) activation in the regulation of hepatic LD formation induced by CLA isomers [cis-9,trans-11 (c9,t11), trans-10,cis-12 (t10,c12)] and linoleic acid (LA) in an in vitro model of lipid accumulation. Dose response of c9,t11 and t10,c12 CLA isomers as well as LA in quiescent H4IIE cells was assessed by Oil Red O staining and subsequent quantification after 24 hours. LD formation was induced by the CLA isomers similar to LA in a dose-dependent manner. However, treatment with the acyl CoA synthetase (ACS) inhibitor, triacsin C, resulted in significantly reduced LD formation. A similar reduction in lipid accumulation was observed with the PPARα activator, Wy14643. Furthermore, CLA isomers promoted H4IIE viability at 60 µM but decreased viability at a higher dose of 180 µM. To further understand the role of PPARα in hepatic steatosis, we studied the level and phosphorylation of PPARα in livers of male lean and fa/fa Zucker rats fed either a control diet or fa/fa Zucker rats fed a CLA isomer (0.4% wt/wt c9,t11 or 0.4% wt/wt t10,c12) diet for 8 weeks. Immunoblotting results showed that only the t10,c12 CLA isomer significantly reduced phospho-PPARα S21 compared to the lean control (ln Ctl) and it was associated with a significant increase in the phosphorylation of p38 mitogen activated protein kinase (MAPK).These changes were not observed with the c9,t11 CLA isomer. Taken together, we have shown that CLA isomers directly induce LD formation in quiescent H4IIEs by activation of the lipid storage pathway which was significantly reduced by triacsin C or Wy14643. Also, we demonstrate for the first time that only the t10,c12 CLA isomer significantly reduced PPARα phosphorylation while it increased p38 MAPK phosphorylation. These results indicate that the anti-steatotic effects of the t10,c12 CLA isomer is associated with changes in PPARα phosphorylation and thereby its activity in a MAPK-independent manner.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.