Transferrin Uptake Research Articles

The role of glycosylphosphatidylinositol phospholipase C in membrane trafficking in Trypanosoma brucei

Glycosylphosphatidylinositol-phospholipase C (GPI-PLC) is an enzyme that has been implicated in GPI-dependent protein trafficking and phosphoinositide metabolism in the bloodstream stage of African trypanosomes. However, despite the fact that it is associated with the cytoplasmic face of internal organellar compartments, its role in general membrane trafficking has not been investigated. Using a GPI-PLC null cell line, we determine the effect of GPI-PLC deficiency on these processes. Biosynthetic trafficking of lysosomal cargo, soluble cathepsin L and membrane bound p67, are unaffected. Likewise, secretory transport, recycling and ultimate lysosomal turnover of the GPI-anchored and transmembrane glycoproteins, transferrin receptor and invariant surface glycoprotein 65, respectively, were unaffected. A significant decrease in the endocytic uptake of transferrin was observed, confirming a prior report, but ultimate delivery to the lysosome was unimpacted. These results contribute to our understanding of the roles of this enigmatic enzyme in trypanosome cell biology.

Nitric oxide may regulate focal adhesion turnover and cell migration in MDA-MB-231 breast cancer cells by modulating early endosome trafficking

Abstract Cell migration is an essential process for wound healing, metastasis and inflammation. Focal adhesions (FA) are local regions of plasma membrane consisting of multiprotein complexes providing adhesive contact between the cell and the extracellular matrix (ECM). FA turnover regulates different signalling pathways implicated in various cellular responses (e.g. cell migration). Endocytosis, specifically the dynamin and clathrin pathways, is known to regulate cell migration by modulating FA dynamics. In this study, we investigated whether NO activity regulates cell migration, FA dynamics and early endosome trafficking in MDA-MB-231 cells. The assessment of cell migration showed a slowing down of cell migration and an increased duration of FA turnover in cells treated with inhibitors of NO synthase (NOS) such as L-NAME or 1400W. In addition, these treatments were found to exhibit no effect on transferrin and dextran uptake mediated by endocytosis and micropinocytosis, respectively. The number of early endosome antigen 1 (EEA1)-positive endosomes was reduced while their sizes were found to increase in cells treated with L-NAME or 1400W. In contrast, these inhibitors did not affect the number nor the size of Rab5-positive endosomes. Furthermore, we demonstrated that EEA1, endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) were colocalised. Using the biotin switch assay followed by western blot, we showed that early endosome proteins such as APPL1, EEA1, Rab5 were found to be S-nitrosylated. These results were further supported by the sequence analysis performed with the GPS-SNO algorithm which predicted the S-nitrosylation of these endosomal proteins. Taken together, our findings suggest that NO might be involved in cell migration and FA turnover through early endosome trafficking in MDA-MB-231 cells. Running title: Nitric oxide in MDA-MB-231 breast cancer cells

NDR1/2 Kinases Enhance Autophagy and Prevent Neurodegeneration via ATG9A Trafficking

NDR kinases NDR1 and NDR2 are highly conserved regulators of neuronal differentiation that are also implicated in mitochondrial health and autophagy. The roles of NDR1/2 in mammalian brain homeostasis in vivo have not been explored. Using constitutive Ndr1 and/or neuron-specific Ndr2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 causes neurodegeneration with progressive accumulation of p62 and ubiquitinated proteins. Consistent with impaired autophagy, we found reduced LC3-positive autophagosomes in the brain and lower autophagosome formation rates and axonal LC3 trafficking in primary neurons expressing NDR1/2 shRNA. Quantitative proteomics revealed altered regulation of membrane trafficking and identified the endocytic protein Raph1/Lamellipodin (Lpd) as a novel substrate of NDR kinases. Pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, partially at synapses, impaired axonal ATG9A trafficking and increased ATG9A surface levels reflect defective endocytosis, as shown by reduced transferrin uptake. We provide mechanistic understanding of how NDR kinases maintain neuronal autophagy and health.

Bi‐allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis‐like disease

Lysosomal storage diseases, including mucopolysaccharidoses, result from genetic defects that impair lysosomal catabolism. Here, we describe two patients from two independent families presenting with progressive psychomotor regression, delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and mucopolysaccharidosis‐like dysmorphic features. Both patients were homozygous for the same intronic variant in VPS16, a gene encoding a subunit of the HOPS and CORVET complexes. The variant impaired normal mRNA splicing and led to an ~85% reduction in VPS16 protein levels in patient‐derived fibroblasts. Levels of other HOPS/CORVET subunits, including VPS33A, were similarly reduced, but restored upon re‐expression of VPS16. Patient‐derived fibroblasts showed defects in the uptake and endosomal trafficking of transferrin as well as accumulation of autophagosomes and lysosomal compartments. Re‐expression of VPS16 rescued the cellular phenotypes. Zebrafish with disrupted vps16 expression showed impaired development, reduced myelination, and a similar accumulation of lysosomes and autophagosomes in the brain, particularly in glia cells. This disorder resembles previously reported patients with mutations in VPS33A, thus expanding the family of mucopolysaccharidosis‐like diseases that result from mutations in HOPS/CORVET subunits.

A genome-wide atlas of co-essential modules assigns function to uncharacterized genes.

A central question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into 'co-essential' pathways, but this approach has been limited by ubiquitous false positives. In the present study, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This atlas recapitulates diverse pathways and protein complexes, and predicts the functions of 108 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, a key enzyme for plasmalogen synthesis. We also show that C15orf57 encodes a protein that binds the AP2 complex, localizes to clathrin-coated pits and enables efficient transferrin uptake. Finally, we provide an interactive webtool for the community to explore our results, which establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of new gene functions.

Sterols Lower Energetic Barriers of Membrane Bending and Fission Necessary for Efficient Clathrin Mediated Endocytosis

As the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

Trypanosoma brucei transferrin receptor: Functional replacement of the GPI anchor with a transmembrane domain

The transferrin receptor of Trypanosoma brucei (TbTfR) is a heterodimer of a glycosylphosphatidylinositol (GPI)-anchored ESAG6 subunit and an ESAG7 subunit. To investigate whether the GPI-anchor is essential for the function of the TbTfR, an ESAG6 with a transmembrane domain instead of a GPI-anchor (ESAG6tmd) was inducibly expressed in bloodstream form trypanosomes. It is shown that the ESAG6tmd is able to dimerise with ESAG7 to form a TbTfR that can bind transferrin. Fractionation experiments clearly demonstrated that the transmembrane-anchored TbTfR is exclusively associated with the membrane fraction. No difference in the uptake of transferrin was observed between trypanosomes inducibly expressing a transmembrane-anchored TbTfR and trypanosomes inducibly expressing a GPI-anchored TbTfR. Differences in glycosylation pattern of ESAG6tmd and native ESAG6 may indicate different intracellular trafficking of transmembrane- and GPI-anchored TbTfRs. The findings suggest that the GPI-anchor is not essential for the function of the TbTfR in bloodstream forms of T. brucei.

Proton-activated chloride channel PAC regulates endosomal acidification and transferrin receptor-mediated endocytosis

SUMMARYDuring vesicular acidification, chloride (Cl−), as the counterion, provides the electrical shunt for proton pumping by the vacuolar H+ ATPase. Intracellular CLC transporters mediate Cl− influx to the endolysosomes through their 2Cl−/H+ exchange activity. However, whole-endolysosomal patch-clamp recording also revealed a mysterious conductance releasing Cl− from the lumen. It remains unknown whether CLCs or other Cl− channels are responsible for this activity. Here, we show that the newly identified proton-activated Cl− (PAC) channel traffics from the plasma membrane to endosomes via the classical YxxL motif. PAC deletion abolishes the endosomal Cl− conductance, raises luminal Cl− level, lowers luminal pH, and increases transferrin receptor-mediated endocytosis. PAC overexpression generates a large endosomal Cl− current with properties similar to those of endogenous conductance, hypo-acidifies endosomal pH, and reduces transferrin uptake. We propose that the endosomal Cl− PAC channel functions as a low pH sensor and prevents hyper-acidification by releasing Cl− from the lumen.

Transferrin receptor 1 is a cellular receptor for human heme-albumin

Iron is essential for living cells. Uptake of iron-loaded transferrin by the transferrin receptor 1 (CD71, TFR) is a major but not sufficient mechanism and an alternative iron-loaded ligand for CD71 has been assumed. Here, we demonstrate that CD71 utilizes heme-albumin as cargo to transport iron into human cells. Binding and endocytosis of heme-albumin via CD71 was sufficient to promote proliferation of various cell types in the absence of transferrin. Growth and differentiation of cells induced by heme-albumin was dependent on heme-oxygenase 1 (HO-1) function and was accompanied with an increase of the intracellular labile iron pool (LIP). Import of heme-albumin via CD71 was further found to contribute to the efficacy of albumin-based drugs such as the chemotherapeutic Abraxane. Thus, heme-albumin/CD71 interaction is a novel route to transport nutrients or drugs into cells and adds to the emerging function of CD71 as a scavenger receptor.

Hypomorphic CAMKK2 in EA.hy926 endothelial cells causes abnormal transferrin trafficking, iron homeostasis and glucose metabolism

We recently reported that loss of calcium/calmodulin-dependent protein kinase kinase-2 (CAMKK2), a serine/threonine kinase activated by intracellular calcium, in mice leads to tissue-specific aberrant turnover of transferrin (TF), a receptor-mediated iron-transporter that supplies iron to tissues. Iron dyshomeostasis is associated with the pathogenesis of several diseases, making TF transport relevant to health. In this study, we used hemizygous CAMKK2 hypomorphic human endothelial cell (EA.hy926) clones to demonstrate that cells with reduced CAMKK2 exhibit increased TF uptake and transcytosis, and decreased intracellular trafficking to subcellular organelles compared to wild-type. The abnormal TF trafficking in CAMKK2 hypomorphic cells correlated with a reduction in intracellular iron content and defective glucose metabolism including glycolysis and mitochondrial respiration. CAMKK2 deficiency also caused reduction in GAPDH and VDAC1 protein level which correlated to defective bioenergetics function. These findings have identified a novel mechanistic link between abnormal calcium signaling, iron dyshomeostasis and metabolic dysfunction involving CAMKK2.

Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1.

LZTFL1 participates in immune synapse formation, ciliogenesis, and the localization of ciliary proteins, and knockout of LZTFL1 induces abnormal distribution of heterotetrameric adaptor protein complex-1 (AP-1) in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 is involved in intracellular transport. Here, we demonstrate that in vitro LZTFL1 directly binds to AP-1 and AP-2 and coimmunoprecipitates AP-1 and AP-2 from cell lysates. DxxFxxLxxxR motif of LZTFL1 is essential for these bindings, suggesting LZTFL1 has roles in AP-1 and AP-2-mediated protein trafficking. Since AP-1 and AP-2 are known to be involved in transferrin receptor 1 (TfR1) trafficking, the effect of LZTFL1 on TfR1 recycling was analyzed. TfR1, AP-1 and LZTFL1 from cell lysates could be coimmunoprecipitated. However, pull-down results indicate there is no direct interaction between TfR1 and LZTFL1, suggesting that LZTFL1 interaction with TfR1 is indirect through AP-1. We report the colocalization of LZTFL1 and AP-1, AP-1 and TfR1 as well as LZTFL1 and TfR1 in the perinuclear region (PNR) and the cytoplasm, suggesting a potential complex between LZTFL1, AP-1 and TfR1. The results from the disruption of adaptin recruitment with brefeldin A treatment suggested ADP-ribosylation factor-dependent localization of LZFL1 and AP-1 in the PNR. Knockdown of AP-1 reduces the level of LZTFL1 in the PNR, suggesting that AP-1 plays a role in LZTFL1 trafficking. Knockout of LZTFL1 reduces the cell surface level and the rate of internalization of TfR1, leading to a decrease of transferrin uptake, efflux, and internalization. However, knockout of LZTFL1 did not affect the cell surface levels of epidermal growth factor receptor and cation-independent mannose 6-phosphate receptor, indicating that LZTFL1 specifically regulates the cell surface level of TfR1. These data support a novel role of LZTFL1 in regulating the cell surface TfR1 level by interacting with AP-1 and AP-2.

Cellular Assays for Measuring Dynamin Activity in Muscle Cells.

Dynamin is one of the best-studied membrane fission machineries, which mediates endocytic vesicle pinch-off from the plasma membrane. Among the three dynamin isoforms encoded in mammalian genome, dynamin-2 is the ubiquitously expressed isoform and leads to human muscular or neuronal diseases when mutants causing hyperactivity or hypoactivity of its membrane fission activity occur. While transferrin uptake is the most commonly used assay to measure dynamin activity in cultured cells, here we provide two different methods to quantitatively examine the activity of dynamin in myoblasts and myotubes, i.e., Bin1-tubule vesiculation and glucose transporter 4 fractionation assays, respectively. These methods could provide a quantitative measurement of dynamin activity in both differentiated and undifferentiated myoblasts.

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Posaconazole is a novel inhibitor for alphavirus viral entry

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes millions of infection globally. Posaconazole (PCZ) is an antifungal drug, which we and others have previously found to inhibit replication of a number of viruses, including dengue virus, a member of the Flaviviridae family. In this study, we analyzed the antiviral activity of PCZ against alphaviruses. We found that PCZ potently inhibits a number of alphaviruses, including Semliki forest virus (SFV), Sindbis virus (SINV) and CHIKV with half maximal effective concentration (EC50) of 2.3 μM, 4.0 μM and 0.8 μM, respectively. Time-of-addition assays indicated that PCZ treatment before and at the time of SFV infection showed potent inhibition, whereas addition of PCZ at later time points post infection showed minor to no inhibition, suggesting inhibition at an early stage of the replication cycle. In accordance, PCZ treatment of a temperature sensitive mutant of SFV that is capable of cell entry and translation, but not RNA replication, resulted in an almost 90% reduction in luciferase activity. To confirm these findings, PCZ resistant mutant virus were generated and we identified mutations in E1 (V148A) and E2 (H255R) viral glycoproteins, of which the E2 mutation confers partial resistance to PCZ when introduced into wild-type SFV. To see whether PCZ alters clathrin-mediated endocytosis, we analyzed the uptake of fluorescence-tagged transferrin and found that PCZ reduced transferrin uptake by 50% compared to DMSO-treated cells. Together, these results establish PCZ as a novel inhibitor of alphaviruses and identify viral entry as its target.

Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane.

T cell activation is immediately followed by internalization of the T cell receptor (TCR). TCR endocytosis is required for T cell activation, but the mechanisms supporting removal of TCR from the cell surface remain incompletely understood. Here we report that TCR endocytosis is linked to the clathrin-independent carrier (CLIC) and GPI-enriched endocytic compartments (GEEC) endocytic pathway. We show that unlike the canonical clathrin cargo transferrin or the adaptor protein Lat, internalized TCR accumulates in tubules shaped by the small GTPase Cdc42 and the Bin/amphiphysin/Rvs (BAR) domain containing protein GRAF1 in T cells. Preventing GRAF1-positive tubules to mature into endocytic vesicles by expressing a constitutively active Cdc42 impairs the endocytosis of TCR, while having no consequence on the uptake of transferrin. Together, our data reveal a link between TCR internalization and the CLIC/GEEC endocytic route supported by Cdc42 and GRAF1.

Structure of the trypanosome transferrin receptor reveals mechanisms of ligand recognition and immune evasion.

To maintain prolonged infection of mammals, African trypanosomes have evolved remarkable surface coats and a system of antigenic variation1. Within these coats are receptors for macromolecular nutrients such as transferrin2,3. These must be accessible to their ligands but must not confer susceptibility to immunoglobulin-mediated attack. Trypanosomes have a wide host range and their receptors must also bind ligands from diverse species. To understand how these requirements are achieved, in the context of transferrin uptake, we determined the structure of a Trypanosoma brucei transferrin receptor in complex with human transferrin, showing how this heterodimeric receptor presents a large asymmetric ligand-binding platform. The trypanosome genome contains a family of around fourteen transferrin receptors4, which has been proposed to allow binding to transferrin from different mammalian hosts5,6. However, we find that a single receptor can bind transferrin from a broad range of mammals, indicating that receptor variation is unlikely to be necessary for promiscuity of host infection. In contrast, polymorphic sites and N-linked glycans are preferentially found in exposed positions on the receptor surface, not contacting transferrin, suggesting that transferrin receptor diversification is driven by a need for antigenic variation in the receptor to prolonged survival in a host.

Expression of a specific variant surface glycoprotein has a major impact on suramin sensitivity and endocytosis in Trypanosoma brucei.

Suramin was introduced into the clinic a century ago and is still used to treat the first stage of acute human sleeping sickness. Due to its size and sixfold negative charge, uptake is mediated through endocytosis and the suramin receptor in trypanosomes is thought to be the invariant surface glycoprotein 75 (ISG75). Nevertheless, we recently identified a variant surface glycoprotein (VSGSur) that confers strong in vitro resistance to suramin in a Trypanosoma brucei rhodesiense line. In this study, we introduced VSGSur into the active bloodstream expression site of a T. b. brucei line. This caused suramin resistance and cross resistance to trypan blue. We quantified the endocytosis of different substrates by flow cytometry and showed that the expression of VSGSur strongly impairs the uptake of low‐density lipoprotein (LDL) and transferrin, both imported by receptor‐mediated endocytosis. However, bulk endocytosis and endocytosis of the trypanolytic factor were not affected, and the VSGSur‐expressors did not exhibit a growth phenotype in the absence of suramin. Knockdown of ISG75 was synergistic with VSGSur expression, indicating that these two proteins are mediating distinct suramin resistance pathways. In conclusion, VSGSur causes suramin resistance in T. brucei bloodstream forms by decreasing specific, receptor‐mediated endocytosis pathways.

IRR is involved in glucose-induced endocytosis after insulin secretion

Endocytosis after insulin secretion plays a pivotal role in the regulation of insulin secretion in pancreatic β-cells. Our recent study suggested that EPI64, a GTPase activating protein for Rab27a, contributes to the regulation of glucose-induced endocytosis, which is mediated by the GDP-bound form of Rab27a. Here, we identified insulin receptor-related receptor (IRR) as an EPI64-interacting protein. Knockdown of IRR inhibited glucose-induced uptake of transferrin, a marker of endocytosis, translocation of the guanine-nucleotide-exchange factor ARNO to the plasma membrane, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). These results suggest that IRR functions upstream of PIP3 generation and controls endocytosis after insulin secretion.

Protein interacting with Amyloid Precursor Protein tail-1 (PAT1) is involved in early endocytosis.

Protein interacting with Amyloid Precursor Protein (APP) tail 1 (PAT1) also called APPBP2 or Ara 67 has different targets such as APP or androgen receptor and is expressed in several tissues. PAT1 is known to be involved in the subcellular trafficking of its targets. We previously observed in primary neurons that PAT1 is poorly associated with APP at the cell surface. Here we show that PAT1 colocalizes with vesicles close to the cell surface labeled with Rab5, Rab4, EEA1 and Rabaptin-5 but not with Rab11 and Rab7. Moreover, PAT1 expression regulates the number of EEA1 and Rab5 vesicles, and endocytosis/recycling of the transferrin receptor. In addition, low levels of PAT1 decrease the size of transferrin-colocalized EEA1 vesicles with time following transferrin uptake. Finally, overexpression of the APP binding domain to PAT1 is sufficient to compromise endocytosis. Altogether, these data suggest that PAT1 is a new actor in transferrin early endocytosis. Whether this new function of PAT1 may have consequences in pathology remains to be determined.

Heterogeneity of Transferrin receptor expression, distribution and transferrin uptake in breast cancer cells in 2D and 3D spheroid cultures

Alterations in the expression of proteins involved in iron homeostasis have been detected in cancer cells, and their levels appear to correlate inversely with patient survival. Thus, elucidating the mechanisms underlying defective iron transport is crucial to understand the faulty iron homeostasis that is typical of cancer cells. Rapidly proliferating cancer cells show increased expression of proteins that support iron import, ex. transferrin receptor (TfR), as well as decreased levels of proteins involved in iron export, ex. ferroportin. TfR (CD71 or p90) delivers iron into cells via the binding of iron‐loaded transferrin (holo‐Tf) and subsequent endocytosis and recycling of TfR‐Tf complexes. Tf is a commonly used ligand for receptor mediated drug delivery and molecular imaging of breast tumors. However, TfR expression and Tf uptake tumor heterogeneity (Figure 1) is a crucial issue that limits our understanding of the role of defective iron transport in tumor progression as well as the use of Tf in targeted drug delivery and tumor imaging. Our long‐term goal is to develop novel imaging methodologies and biochemical approaches to establish, quantitate and modify TfR expression and Tf uptake heterogeneity using tumor xenografts and 3D cancer spheroids (Figure 1). Herein, novel approaches to modulate Tf uptake will be tested for their effect on the heterogeneity of Tf delivery across solid tumors. Previously, increasing the serum concentration of iron‐deleted Tf (apo‐Tf) has been shown to alter Tf saturation as well as the intracellular distribution and level of TfR expression in mice models of anemia, e.g. β‐Thalassemia. We currently hypothesize that increasing concentration of apo‐Tf will lead to a re‐distribution of TfR to the surface of the cells, as well as altered trafficking of iron in cancer cells. We will test the effect of apo‐Tf in TfR expression and distribution in human breast cancer 2D cell cultures, 3D cancer spheroids and tumor xenografts. Firstly, Tf uptake plate reader assays have shown that 24h incubation with apo‐Tf modulates Tf uptake differently in non‐cancerous breast epithelial cells (MCF10A) and in human breast cancer cells (MDA‐MB‐231 and T47D cells). Immunofluorescence imaging of non‐cancerous vs. breast cancer cells has shown that apo‐Tf treatment induces significant changes in the intracellular distribution of TfR and early endosomal marker EEA1. These results suggest that treatment with apo‐Tf acts via alteration of TfR‐Tf endocytic trafficking pathway. Similar experiments will be performed to determine how incubation with apo‐Tf affects TfR expression and Tf uptake heterogeneity in 3D cancer spheroids. Ability to potentially alter TfR trafficking in breast cancer cells is highly significant not only to alter iron homeostasis as a way to reduce cancer progression but also to optimize Tf‐mediated drug delivery approaches. Funding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.Support or Funding InformationFunding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.