Apicobasal transferrin receptor localization and trafficking in brain capillary endothelial cells.

The detailed mechanisms by which the transferrin receptor (TfR) and associated ligands traffic across brain capillary endothelial cells (BECs) of the CNS-protective blood-brain barrier constitute an important knowledge gap within maintenance and regulation of brain iron homeostasis. This knowledge gap also presents a major obstacle in research aiming to develop strategies for efficient receptor-mediated drug delivery to the brain. While TfR-mediated trafficking from blood to brain have been widely studied, investigation of TfR-mediated trafficking from brain to blood has been limited. In this study we investigated TfR distribution on the apical and basal plasma membranes of BECs using expansion microscopy, enabling sufficient resolution to separate the cellular plasma membranes of these morphological flat cells, and verifying both apical and basal TfR membrane domain localization. Using immunofluorescence-based transcellular transport studies, we delineated endosomal sorting of TfR endocytosed from the apical and basal membrane, respectively, as well as bi-directional TfR transcellular transport capability. The findings indicate different intracellular sorting mechanisms of TfR, depending on the apicobasal trafficking direction across the BBB, with the highest transcytosis capacity in the brain-to-blood direction. These results are of high importance for the current understanding of brain iron homeostasis. Also, the high level of TfR trafficking from the basal to apical membrane of BECs potentially explains the low transcytosis which are observed for the TfR-targeted therapeutics to the brain parenchyma.

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers.

Parkinson's disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson's disease and related synucleinopathies.

Regulated exocytosis: renal aquaporin-2 3D vesicular network organization and association with F-actin.

Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes, including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 (AQP2) localizes to the apical plasma membrane as well as to small, subapical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2-containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nanoscale size of these vesicles has limited analysis of their three-dimensional (3D) organization. Using a cell system combined with 3D superresolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2-containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 ± 3 nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the subcortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association were enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis.

Mapping Receptor Antibody Endocytosis and Trafficking in Brain Endothelial Cells.

Drug delivery to the brain is a tremendous problem for the academic society and the industry. One solution with a huge potential is to use endocytic receptors as carriers. Here we describe how endocytic activity and subcellular trafficking of a specific receptor in brain endothelial cells can be characterized in three steps. (1) Labeling, endocytosis, and trafficking of a specific receptor at given time points in a pulse-chase experiment. (2) Fixed antibody labeling and co-staining of subcellular markers for image acquisition. (3) Analysis and quantification of co-localization between the receptor and subcellular markers in ImageJ.

Vesicular Transport Machinery in Brain Endothelial Cells: What We Know and What We Do not.

The vesicular transport machinery regulates numerous essential functions in cells such as cell polarity, signaling pathways, and the transport of receptors and their cargoes. From a pharmaceutical perspective, vesicular transport offers avenues to facilitate the uptake of therapeutic agents into cells and across cellular barriers. In order to improve receptor-mediated transcytosis of biologics across the blood-brain barrier and into the diseased brain, a detailed understanding of intracellular transport mechanisms is essential. The vesicular transport machinery is a highly complex network and involves an array of protein complexes, cytosolic adaptor proteins, and the subcellular structures of the endo-lysosomal system. The endo-lysosomal system includes several types of vesicular entities such as early, late, and recycling endosomes, exosomes, ectosomes, retromer-coated vesicles, lysosomes, trans-endothelial channels, and tubules. While extensive research has been done on the trafficking system in many cell types, little is known about vesicular trafficking in brain endothelial cells. Consequently, assumptions on the transport system in endothelial cells are based on findings in polarised epithelial cells, although recent studies have highlighted differences in the endothelial system. This review highlights aspects of the vesicular trafficking machinery in brain endothelial cells, including recent findings, limitations, and opportunities for further studies.

Vasopressin-Independent Regulation of Aquaporin-2 by Tamoxifen in Kidney Collecting Ducts.

Arginine vasopressin (AVP) mediates water reabsorption in the kidney collecting ducts through regulation of aquaporin-2 (AQP2). Also, estrogen has been known to regulate AQP2. Consistently, we previously demonstrated that tamoxifen (TAM), a selective estrogen receptor modulator, attenuates the downregulation of AQP2 in lithium-induced nephrogenic diabetes insipidus (NDI). In this study, we investigated the AVP-independent regulation of AQP2 by TAM and the therapeutic effect of TAM on the dysregulation of AQP2 and impaired urinary concentration in a unilateral ureteral obstruction (UUO) model. Primary cultured inner medullary collecting duct (IMCD) cells from kidneys of male Sprague-Dawley rats were treated with TAM. Rats subjected to 7 days of UUO were treated with TAM by oral gavage. Changes of intracellular trafficking and expression of AQP2 were evaluated by quantitative PCR, Western blotting, and immunohistochemistry. TAM induced AQP2 protein expression and intracellular trafficking in primary cultured IMCD cells, which were independent of the vasopressin V2 receptor (V2R) and cAMP activation, the critical pathways involved in AVP-stimulated regulation of AQP2. TAM attenuated the downregulation of AQP2 in TGF-ß treated IMCD cells and IMCD suspensions prepared from UUO rats. TAM administration in vivo attenuated the downregulation of AQP2, associated with an improvement of urinary concentration in UUO rats. In addition, TAM increased CaMKII expression, suggesting that calmodulin signaling pathway is likely to be involved in the TAM-mediated AQP2 regulation. In conclusion, TAM is involved in AQP2 regulation in a vasopressin-independent manner and improves urinary concentration by attenuating the downregulation of AQP2 and maintaining intracellular trafficking in UUO.

A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging.

Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.

Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore.

Endocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.

Ectopic expression of aquaporin-5 in noncancerous epithelial MDCK cells changes cellular morphology and actin fiber formation without inducing epithelial-to-mesenchymal transition.

Aquaporin-5 (AQP5) is a plasma membrane water channel mainly expressed in secretory glands. Increased expression of AQP5 is observed in multiple cancers, including breast cancer, where high expression correlates with the degree of metastasis and poor prognosis. Moreover, studies in cancer cells have suggested that AQP5 activates Ras signaling, drives morphological changes, and in particular increased invasiveness. To design intervention strategies, it is of utmost importance to characterize and dissect the cell biological changes induced by altered AQP5 expression. To isolate the effect of AQP5 overexpression from the cancer background, AQP5 was overexpressed in normal epithelial MDCK cells which have no endogenous AQP5 expression. AQP5 overexpression promoted actin stress fiber formation and lamellipodia dynamics. Moreover, AQP5 decreased cell circularity. Phosphorylation of AQP5 on serine 156 in the second intracellular loop has been shown to activate the Ras pathway. When serine 156 was mutated to alanine to mimic the nonphosphorylated state, the decrease in cell circularity was reversed, indicating that the AQP5-Ras axis is involved in the effect on cell shape. Interestingly, the cellular changes mediated by AQP5 were not associated with induction of epithelial-to-mesenchymal transition. Thus, AQP5 may contribute to cancer by altering cellular morphology and actin organization, which increase the metastatic potential.

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature.

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.

Endocytic turnover of Rab8 controls cell polarization.

Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.

Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1.

Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae.

Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis owing to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 (also known as PRKCDBP) is targeted to caveolae by cavin1 (also known as PTRF) where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 (also known as SDPR) region, showing that the cavins form distinct subcomplexes through their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae that are only present at the membrane for a short time. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

Differential roles of Grb2 and AP-2 in p38 MAPK- and EGF-induced EGFR internalization.

The epidermal growth factor receptor (EGFR) is an important regulator of normal growth and differentiation, and it is involved in the pathogenesis of many cancers. Endocytic downregulation is central in terminating EGFR signaling after ligand stimulation. It has been shown that p38 MAPK activation also can induce EGFR endocytosis. This endocytosis lacks many of the characteristics of ligand-induced EGFR endocytosis. We compared the two types of endocytosis with regard to the requirements for proteins in the internalization machinery. Both types of endocytosis require clathrin, but while epidermal growth factor (EGF)-induced EGFR internalization also required Grb2, p38 MAPK-induced internalization did not. Interestingly, AP-2 knock down blocked p38 MAPK-induced EGFR internalization, but only mildly affected EGF-induced internalization. In line with this, simultaneously mutating two AP-2 interaction sites in EGFR affected p38 MAPK-induced internalization much more than EGF-induced EGFR internalization. Thus, it seems that EGFR in the two situations uses different sets of internalization mechanisms.