Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly ...characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P2‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P2 generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P2 in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P2 exchange between late and recycling endosomes.
Synopsis
How LDL‐cholesterol is delivered from late endosomes to the plasma membrane and enhances cell motility is unknown. Here, this is found to depend on ORP2‐mediated lipid exchange between late endosomes and focal adhesion kinase (FAK)‐positive recycling endosomes promoting FAK activation.
Loss of ORP2 abrogates LDL‐cholesterol delivery from late to recycling endosomes.
LDL‐cholesterol activates FAK pending ORP2‐mediated cholesterol transfer.
FAK activity promotes PI(4,5)P2 generation in endosomes, fueling the delivery of LDL‐cholesterol to the plasma membrane.
ORP2 controls endomembrane distribution of PI(4,5)P2 and tubulation of NPC1‐positive endosomes.
Cholesterol effects on cell motility depend on ORP2‐mediated lipid transfer from late endosomes to focal adhesion kinase‐/integrin‐positive recycling endosomes.
The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that ...traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol
like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.
Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, ...especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.
Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 ...mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.
Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have ...remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC- and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a- and Myosin5b (Myo5b)-dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement.
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•Rab8a-interactome regulates postendosomal LDL cholesterol transport•Rabin8, TBC1D30, and Myo5b contribute to the endosomal egress of cholesterol•CD63-containing lysosome-related organelles transport cholesterol to the PM•LDL-derived cholesterol modulates focal adhesions and promotes cell migration
How does LDL cholesterol reach the plasma membrane from endolysosomal organelles? Kanerva et al. elucidate a pathway comprising Rab8a and Rab8-interacting factors that regulates LDL cholesterol transport downstream of NPC1. This route targets cholesterol to focal adhesions via lysosome-related organelles and promotes cell movement by regulating cell adhesion dynamics.
Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was ...found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this issue of Developmental Cell, Marques et al. (2019) provide evidence that the main receptor for high-density lipoporoteins (HDL), scavenger receptor B1 (SR-B1), forms large and dynamic ...homo-multimers at the plasma membrane. This helps the receptor to evade endocytosis, bind HDL, and facilitate selective lipid uptake from HDL.
In this issue of Developmental Cell, Marques et al. (2019) provide evidence that the main receptor for high-density lipoporoteins (HDL), scavenger receptor B1 (SR-B1), forms large and dynamic homo-multimers at the plasma membrane. This helps the receptor to evade endocytosis, bind HDL, and facilitate selective lipid uptake from HDL.
Upon IgE-mediated activation, mast cells (MC) exocytose their cytoplasmic secretory granules and release a variety of bioactive substances that trigger inflammatory responses. Polyamines mediate ...numerous cellular and physiological functions. We report here that MCs express antizyme inhibitor 2 (AZIN2), an activator of polyamine biosynthesis, previously reported to be exclusively expressed in the brain and testis. We have investigated the intracellular localization of AZIN2 both in resting and activated MCs. In addition, we have examined the functional role of polyamines, downstream effectors of AZIN2, as potential regulators of MC activity.
Immunostainings show that AZIN2 is expressed in primary and neoplastic human and rodent MCs. We demonstrate that AZIN2 localizes in the Vamp-8 positive, serotonin-containing subset of MC granules, but not in tryptase-containing granules, as revealed by double immunofluorescence stainings. Furthermore, activation of MCs induces rapid upregulation of AZIN2 expression and its redistribution, suggesting a role for AZIN2 in secretory granule exocytosis. We also demonstrate that release of serotonin from activated MCs is polyamine-dependent whereas release of histamine and beta-hexosaminidase is not, indicating a granule subtype-specific function for polyamines.
The study reports for the first time the expression of AZIN2 outside the brain and testis, and demonstrates the intracellular localization of endogenous AZIN2 in MCs. The granule subtype-specific expression and its induction after MC activation suggest a role for AZIN2 as a local, in situ regulator of polyamine biosynthesis in association with serotonin-containing granules of MCs. Furthermore, our data indicates a novel function for polyamines as selective regulators of serotonin release from MCs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are ...poorly characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells.
In vitro
, cholesterol enhances membrane association of FAK to PI(4,5)P
2
‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P
2
generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P
2
in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P
2
exchange between late and recycling endosomes.
Synopsis
image
How LDL‐cholesterol is delivered from late endosomes to the plasma membrane and enhances cell motility is unknown. Here, this is found to depend on ORP2‐mediated lipid exchange between late endosomes and focal adhesion kinase (FAK)‐positive recycling endosomes promoting FAK activation.
Loss of ORP2 abrogates LDL‐cholesterol delivery from late to recycling endosomes.
LDL‐cholesterol activates FAK pending ORP2‐mediated cholesterol transfer.
FAK activity promotes PI(4,5)P
2
generation in endosomes, fueling the delivery of LDL‐cholesterol to the plasma membrane.
ORP2 controls endomembrane distribution of PI(4,5)P
2
and tubulation of NPC1‐positive endosomes.
Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in ...the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells.
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