The authors have no acknowledgements.

1. Western Blotting of CNTFR&#945; Lysates
<ol>
	<li>Express full-length sorLA and Myc-tagged CNTFR&#945; constructs in human embryonic kidney 293 (HEK293) cells using pcDNA3.1/zeo(-) and pcDNA3.1/hyg(-), respectively. Transfect the HEK293 cells with the constructs using a commercial transfection reagent according to manufacturer&#180;s protocol. Select stable clones in medium containing 150 &#181;g/mL Zeocin and/or 500 &#181;g/mL Hygromycin B Gold13.</li>
	<li>Seed equal numbers of HEK293 transfectants expressing either CNTFR&#945;-Myc or co-expressing CNTFR&#945;-Myc/sorLA in two 4-well plates as shown in Figure 1. Culture the cells in Dulbecco Modified Eagle&#180;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 &#956;g/mL streptomycin (full medium) in a 5% carbon dioxide incubator at 37 &#730;C.</li>
	<li>Wait until the cells are 50 - 80% confluent.</li>
	<li>Remove the medium from the wells, wash once with Dulbecco Phosphate Buffered Saline (DPBS), and add fresh full medium with or without (control) 10 nM CLC:CLF-1 as indicated in Figure 1. Incubate the cells at 37 &#730;C.</li>
	<li>After 0 and 5 h of incubation, recover the medium and store it at 4 &#730;C in 1.5 mL tubes. Then add 1% Triton X-100 lysis buffer (20 mM Tris-HCl, 10 mM EDTA, pH 8.0) supplemented with a proteinase inhibitor cocktail to each well (1 min, 4 &#730;C). Subsequently, transfer the cell lysates to 1.5 mL tubes (4 &#730;C).</li>
	<li>Spin the tubes containing the medium and cell lysate (3,000 x g, 3 min, 4 &#730;C) and transfer the supernatants to 1.5 mL tubes (4 &#730;C). Discard the pellet. Transfer 5 &#956;L of each cell lysate supernatant to new 1.5 mL tubes &#8212; use them to measure the protein content later (step 1.7). Immediately, add nonreducing LDS sample buffer to all the supernatants and vortex the samples.</li>
	<li>Use the 5 &#956;L aliquots to measure the protein content in the cell lysate supernatants using a commercial protein assay according to manufacturer&#39;s protocol.</li>
	<li>Heat the samples at 95 &#730;C for 5 min.</li>
	<li>Subject an equal amount of protein from each sample to reducing SDS-PAGE13 and Western blotting13 and visualize the content of sorLA, &#946;-actin, and CNTFR&#945;-Myc in the medium and in the cell lysates using rabbit anti-sorLA16 (5 &#956;g/mL), mouse anti-&#946;-actin (0.4 &#956;g/mL), and mouse anti-Myc (1 &#956;g/mL) antibodies and the appropriate HRP-linked secondary antibodies (1:2,000).</li>
	<li>Quantify the bands by densitometry according to manufacturer&#39;s protocol.</li>
</ol>
2. Immunocytochemistry in Combination with Lysosomal Inhibitors
<ol>
	<li>Seed HEK293 cells stably transfected with CNTFR&#945;-Myc/sorLA on cover slides in a 4-well plate as shown in Figure 2. Culture the cells in full medium as above.</li>
	<li>Wait until cell confluence is 20 - 40%.</li>
	<li>Remove the medium from the wells and add full medium with or without 50 &#956;g/mL of leupeptin and of pepstatin A (leu/pep) (inhibits lysosomal hydrolases) to the cells as indicated in Figure 2. Incubate the cells at 37 &#730;C. The medium must be replaced every 6 h during the following 24 h of incubation.</li>
	<li>After 24 h of incubation, once again replace the medium (with or without leu/pep) and this time add 10 nM CLC:CLF-1. Incubate the cells at 37 &#730;C for 5 h.</li>
	<li>Wash the cells once in PBS and fix them in 4% paraformaldehyde, pH 7 for 15 min at RT. 
	Caution: Paraformaldehyde is highly toxic, avoid contact with skin, eyes, and mucous. Gloves and safety glasses should be worn and solutions made inside a fume hood.</li>
	<li>Wash the cells once in PBS and permeabilize the cells for 5 min at RT in saponin diluted in PBS (0.5% saponin).</li>
	<li>Dilute goat anti-CNTFR&#945; (1 &#956;g/mL) (notably not the mouse anti-Myc used for Western blotting) and mouse anti-LAMP-1 (15 &#956;g/mL) antibodies in 0.5% saponin and incubate the cells with the antibodies for 2 h at RT.</li>
	<li>Wash the cells 4x (5 min) in 0.5% saponin and incubate the cells with donkey anti-goat Alexa 488- and donkey anti-mouse Alexa 568-conjugated secondary antibodies (6 &#956;g/mL) for 2 h at RT.</li>
	<li>Wash the cells 4x (5 min) in 0.5% saponin, twice in deionized water (1 min), and mount the cover slides on microscope slides using mounting media.</li>
	<li>Analyze the antibody-staining of the cells by confocal microscopy using a laser-scanning confocal microscope with a 63X C-apochromat water immersion objective, NA 1.2.</li>
</ol>
3. Western Blot-detection of the pSTAT3 Level
<ol>
	<li>Seed HEK293-sorLA cells (which express endogenous levels of CNTFR&#945;) in a 4-well plate as shown in Figure 3. Culture the cells in full medium as above.</li>
	<li>Wait until the cells are 50 - 80% confluent.</li>
	<li>Remove the medium from the wells, wash once in PBS, and add fresh full medium with or without 10 nM CLC:CLF-1 to the cells as indicated in Figure 3. Incubate the cells at 37 &#730;C for 5 h.</li>
	<li>Remove the medium, wash the cells twice in unsupplemented DMEM (blank medium; without FBS and antibiotics), and then reincubate the cells for 90 min (37 &#730;C) in blank medium.</li>
	<li>Once again, remove the medium and add fresh blank medium with or without 5 nM CLC:CLF-1 (to initiate STAT3 phosphorylation) as indicated in Figure 3. Incubate for 15 min at 37 &#730;C.</li>
	<li>Quickly remove the medium and lyse the cells (1 min, 4 &#730;C) by adding 1% Triton X-100 lysis buffer (20 mM Tris-HCl, 10 mM EDTA, pH 8.0) supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail to each well. Subsequently, transfer the cell lysates to 1.5 mL tubes (4 &#730;C).</li>
	<li>Transfer 5 &#956;L of each supernatant to new 1.5 mL tubes &#8212; use them to measure the protein content later (step 3.8). Immediately, add nonreducing LDS sample buffer to all the supernatants and vortex the samples.</li>
	<li>Measure the protein content of the 5 &#956;L aliquots using a commercial protein assay according to manufacturer&#39;s protocol.</li>
	<li>Sonicate each sample at RT until&#160;they are no longer viscous (approximately 1 min) and subsequently heat them at 95 &#730;C for 5 min.</li>
	<li>Subject an equal amount of protein from each sample to SDS-PAGE13 and Western blotting13 and visualize the content of pSTAT3 and total STAT3 in the cell lysates using rabbit anti-pSTAT3 (1:2000) and mouse anti-STAT3 (1:1,000) antibodies and the appropriate HRP-linked mouse and rabbit secondary antibodies (1:2,000).</li>
	<li>Quantify the bands by densitometry according to manufacturer&#39;s protocol.</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-like+factor-1,+a+novel+soluble+protein,+shares+homology+with+members+of+the+cytokine+type+I+receptor+family.">Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.</a> J Immunol. 161 (3), 1371-1379 (1998).</li><li>Lelievre, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+recruited+by+the+cardiotrophin-like+cytokine/cytokine-like+factor-1+composite+cytokine:+specific+requirement+of+the+membrane-bound+form+of+ciliary+neurotrophic+factor+receptor+alpha+component.">Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component.</a> J Biol Chem. 276 (25), 22476-22484 (2001).</li><li>Zou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Death+in+Mice+Lacking+Cardiotrophin-Like+Cytokine+(CLC)+is+Associated+with+Multifocal+Neuronal+Hypoplasia.">Neonatal Death in Mice Lacking Cardiotrophin-Like Cytokine (CLC) is Associated with Multifocal Neuronal Hypoplasia.</a> Vet Pathol. 46 (3), 514-519 (2008).</li><li>Forger, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiotrophin-like+cytokine/cytokine-like+factor+1+is+an+essential+trophic+factor+for+lumbar+and+facial+motoneurons+in+vivo.">Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic factor for lumbar and facial motoneurons in vivo.</a> J Neurosci. 23 (26), 8854-8858 (2003).</li><li>Alexander, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suckling+defect+in+mice+lacking+the+soluble+haemopoietin+receptor+NR6.">Suckling defect in mice lacking the soluble haemopoietin receptor NR6.</a> Curr Biol. 9 (11), 605-608 (1999).</li><li>DeChiara, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+the+CNTF+receptor,+unlike+mice+lacking+CNTF,+exhibit+profound+motor+neuron+deficits+at+birth.">Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth.</a> Cell. 83 (2), 313-322 (1995).</li><li>Crisponi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crisponi+syndrome+is+caused+by+mutations+in+the+CRLF1+gene+and+is+allelic+to+cold-induced+sweating+syndrome+type+1.">Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1.</a> Am J Hum Genet. 80 (5), 971-981 (2007).</li><li>Crisponi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+recessive+disorder+with+muscle+contractions+resembling+neonatal+tetanus,+characteristic+face,+camptodactyly,+hyperthermia,+and+sudden+death:+a+new+syndrome.">Autosomal recessive disorder with muscle contractions resembling neonatal tetanus, characteristic face, camptodactyly, hyperthermia, and sudden death: a new syndrome.</a> Am J Med Genet. 62 (4), 365-371 (1996).</li><li>Sohar, E., Shoenfeld, Y., Udassin, R., Magazanik, A., Revach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-induced+profuse+sweating+on+back+and+chest.+A+new+genetic+entity.">Cold-induced profuse sweating on back and chest. A new genetic entity.</a> Lancet. 2 (8099), 1073-1074 (1978).</li><li>Guillet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+active+fusion+protein+of+the+novel+composite+cytokine+CLC/soluble+CNTF+receptor.">Functionally active fusion protein of the novel composite cytokine CLC/soluble CNTF receptor.</a> Eur J Biochem. 269 (7), 1932-1941 (2002).</li><li>Larsen, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-Like+Factor+1,+an+Essential+Facilitator+of+Cardiotrophin-Like+Cytokine:Ciliary+Neurotrophic+Factor+Receptor+alpha+Signaling+and+sorLA-Mediated+Turnover.">Cytokine-Like Factor 1, an Essential Facilitator of Cardiotrophin-Like Cytokine:Ciliary Neurotrophic Factor Receptor alpha Signaling and sorLA-Mediated Turnover.</a> Mol Cell Biol. 36 (8), 1272-1286 (2016).</li><li>Willnow, T. E., Petersen, C. M., Nykjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VPS10P-domain+receptors+-+regulators+of+neuronal+viability+and+function.">VPS10P-domain receptors - regulators of neuronal viability and function.</a> Nat Rev Neurosci. 9 (12), 899-909 (2008).</li><li>Jacobsen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+a+novel+human+hybrid-type+receptor+that+binds+the+alpha2-macroglobulin+receptor-associated+protein.">Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein.</a> J Biol Chem. 271 (49), 31379-31383 (1996).</li><li>Nielsen, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+by+the+cytoplasmic+domain+of+the+amyloid+precursor+protein+binding+receptor+SorLA.">Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA.</a> Mol Cell Biol. 27 (19), 6842-6851 (2007).</li><li>Jacobsen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sorLA+cytoplasmic+domain+interacts+with+GGA1+and+-2+and+defines+minimum+requirements+for+GGA+binding.">The sorLA cytoplasmic domain interacts with GGA1 and -2 and defines minimum requirements for GGA binding.</a> FEBS Lett. 511 (1-3), 155-158 (2002).</li><li>Klinger, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SorLA+regulates+the+activity+of+lipoprotein+lipase+by+intracellular+trafficking.">SorLA regulates the activity of lipoprotein lipase by intracellular trafficking.</a> J Cell Sci. 124 (7), 1095-1105 (2011).</li><li>Kitago, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+amyloidogenic+peptide+recognition+by+sorLA.">Structural basis for amyloidogenic peptide recognition by sorLA.</a> Nat Struct Mol Biol. 22 (3), 199-206 (2015).</li><li>Quistgaard, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligands+bind+to+Sortilin+in+the+tunnel+of+a+ten-bladed+beta-propeller+domain.">Ligands bind to Sortilin in the tunnel of a ten-bladed beta-propeller domain.</a> Nat Struct Mol Biol. 16 (1), 96-98 (2009).</li><li>Larsen, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+sorCS1+binds+sortilin+and+hampers+its+cellular+functions.">Human sorCS1 binds sortilin and hampers its cellular functions.</a> Biochem J. 457 (2), 277-288 (2014).</li><li>Larsen, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+facilitates+signaling+of+ciliary+neurotrophic+factor+and+related+helical+type+1+cytokines+targeting+the+gp130/leukemia+inhibitory+factor+receptor+beta+heterodimer.">Sortilin facilitates signaling of ciliary neurotrophic factor and related helical type 1 cytokines targeting the gp130/leukemia inhibitory factor receptor beta heterodimer.</a> Mol Cell Biol. 30 (17), 4175-4187 (2010).</li></ol>

The authors have nothing to disclose.

The protocol described here can be used specifically to demonstrate the sorLA-mediated CLC:CLF-1-dependent downregulation and lysosomal targeting of CNTFR&#945;, as well as the accompanying weakened response to CLC:CLF-1 stimulation.
The HEK293 cell line is well-suited for this protocol, as they have only a minor endogenous expression of CNTFR&#945; and sorLA, are easy to transfect, express gp130/LIFR&#946;, and have a large cell body, which is suited for immunocytochemistry. However, in theory any cell line with similar properties should work as well.
The protocol has two critical steps. The first concerns step 2.3, in which the leu/pep medium must be replaced approximately every 6 h for 24 h. Failure to do so may result in active lysosomal enzymes and less or no detectable accumulation of protein in the lysosomes. The second critical step (3.4) concerns washing upon pre-incubation with CLC:CLF-1. It is important to remove any unbound ligand, and to allow the cells time to recover (avoiding continued stimulation) in unsupplemented DMEM (blank medium). This will ensure a low background level of pSTAT3 during the subsequent re-stimulation with CLC:CLF-1 (step 3.5).
Notably, Figure 6 demonstrates that the cellular response (pSTAT3) decreases in parallel with the sorLA-mediated downregulation of CNTFR&#945;. It should be emphasized however, that although a reduced response is in accordance with (and to be expected upon) a reduction of the CNTFR&#945; pool, it does not prove that the low CNTFR&#945; expression alone accounts for the reduced cellular response. Thus, changes &#8212; resulting from pre-stimulation or transfection &#8212; in any of the functional elements of the signaling pathway upstream to pSTAT3 may have contributed to the altered response.
The basic concept of the protocol is not limited to this particular receptor system. By modifying the protocol (i.e. another cell line, other antibodies, other ligands, etc.) it can be used to determine the ligand-induced downregulation of other receptors as well &#8212; regardless of sorLA&#180;s involvement. However, it should be noted that analysis of receptor-downregulation by Western blotting is mainly suited for receptors, e.g., GPI-anchored receptors, which exhibit a slow turnover and a low degree of new synthesis in unstimulated cells. Thus, in receptors showing a high level of new-synthesis, ligand-induced downregulation may be compensated for by synthesis of new receptors. Under these circumstances, the downregulation of the receptor pool can instead be determined by means of metabolic labeling and pulse-chase experiments as described in21. Alternatively, iodinated antibodies (preferably Fc-fragments) that do not interfere with receptor binding can be used to &#34;tag&#34; the ectodomain of the receptor, and the expected downregulation can then be determined by radioactive counting of cell pellet and medium.
For logistic reasons we transfected our cells with a CNTFR&#945; construct carrying a Myc-tag, and in the present protocol a mouse anti-Myc antibody was used for Western blot-detection of the receptor. In contrast, a goat anti-CNTFR&#945; antibody was used for immunocytochemistry and double labeling as LAMP-1 was detected by a mouse antibody &#8212; and obviously use of two primary mouse antibodies would result in unspecific (overlapping) staining with secondary antibodies. Note that since the goat anti-CNTFR&#945; also works in Western blotting (not shown), the protocol could easily be modified and applied to cells transfected with untagged CNTFR&#945;.
Finally, it has recently been shown that also the endocytic receptor sortilin binds CLC:CLF-1 with high affinity22. Thus, it is conceivable that sortilin, like sorLA, interconnects to CLC:CNTFR&#945; via CLF-1 and is able to mediate endocytosis and lysosomal targeting of CNTFR&#945; as well. Using sortilin instead of sorLA, the present protocol may be used to clarify this question.

The CLC and&#160;CLF-1 constitute the two subunits of the heterodimeric cytokine CLC:CLF-11-3. For cellular signal induction, CLC:CLF-1 first targets the CNTFR&#945;, its primary and GPI-anchored receptor, to form a membrane-bound trimeric complex. The CLC:CLF-1:CNTFR&#945; complex subsequently recruits gp130/LIFR&#946; which mediates transmembrane signaling via the Janus Kinase/Signal Transducers and Activators of Transcription 3 (JAK/STAT3) pathway4. Mice deficient in any of the three subunits display one and the same phenotype and die within 24 h after birth due to a reduced number of facial neurons and insufficient suckling5-8. Findings in humans likewise underscore the functional coherence of the three subunits. Thus, human mutations (homozygote or compound) causing dysfunction of CLC:CLF-1 or CNTFR&#945;, all result in the so-called cold-induced sweating/Crisponi syndrome, a condition characterized by symptoms such as impaired suckling and swallowing, various dysmorphic features, temperature spikes, and paradoxical and perfuse sweating at low temperatures9-11.
In vitro, the combination of CLC and CNTFR&#945; is both necessary and sufficient for interaction with gp130/LIFR&#946; and the induction of signaling in cells12. The role of CLF-1 on the other hand is less clear. It is not directly involved in signaling, and has long been regarded as an appendix that mainly serves to facilitate the cellular secretion of CLC1. However, recent findings show that CLF-1 has additional and more important functions implicating both the signaling and turnover of CLC and CNTFR&#945;. Thus, it appears that CLF-1 contains three independent binding sites: one for its well-known binding to CLC; one that mediates direct binding to the CNTFR&#945;; and a third (high affinity) site for interaction with the endocytic receptor sorLA. As both CLC and CLF-1 seem&#160;to target CNTFR&#945; with a considerable lower affinity than the CLC:CLF-1 complex, it is conceivable that CLF-1 (via its CNTFR&#945;-binding site) promotes the unification of CLC and CNTFR&#945; and thereby facilitates signaling13.
CLF-1&#39;s interaction with sorLA, the main issue of the present presentation, plays a completely different role. SorLA is one of the five type 1 receptors that constitute the Vps10p-domain receptor family14. It is expressed in a variety of tissues but in particular in brain and neuronal tissues15. Similar to the other family members sorLA carries an N-terminal ligand binding Vps10p-domain, but in addition, it also comprises other domain types including ligand-binding elements found in members of the low-density lipoprotein receptor family15. Its cytoplasmic tail interacts with several adaptor proteins, e.g., adaptor protein-1 and -2, and sorLA conveys efficient endocytosis as well as intracellular sorting and transport of bound proteins16-18. The Vps10p-domain comprises a large ten-bladed &#946;-propeller19,20, which binds a series of unrelated ligands including CLF-1 (but notably, not CLC)13. The binding site in CLF-1 is accessible even after complex formation with CLC and CNTFR&#945; which means that sorLA binds not only free CLF-1, but also CLC:CLF-1 and the trimeric complex CLC:CLF-1:CNTFR&#945;13. SorLA conveys rapid endocytosis of CLF-1 and CLC:CLF-1, but these are soluble proteins whereas CNTFR&#945; is fixed to the surface membrane by a GPI-anchor. The question is therefore if binding of CLC:CLF-1:CNTFR&#945; allows sorLA to internalize the entire complex and thereby to alter the surface-membrane expression of CNTFR&#945; (and the subsequent cellular susceptibility to CLC:CLF-1 signal induction), and/or the turnover of CNTFR&#945;.
The experiments described in the present report were designed to clarify the following questions:
Does sorLA mediate CLC:CLF-1-dependent downregulation of surface-membrane CNTFR&#945;? To clarify this, it was initially tried to determine the turnover of CNTFR&#945; (in the absence and presence of sorLA) by means of metabolic labeling and pulse-chase experiments as described in21. However, attempts to label CNTFR&#945; using a mixture of [S35] cysteine and [S35] methionine showed poor incorporation of radioactivity. This suggested a very low degree of new-synthesis, which in all probability would be unable to compensate for a sudden and significant loss of receptors. To determine if CNTFR&#945; was downregulated when interconnected via CLC:CLF-1 to sorLA, it was therefore decided simply to measure (using Western blotting) the total cellular pool of CNTFR&#945; before and after exposure to CLC:CLF-1 - and to compare results in cells transfected or not transfected with sorLA.
Does sorLA target CNTFR&#945; to lysosomes? To answer this question, the localization of CNTFR&#945; &#8212; internalized via its CLC:CLF-1-mediated binding to sorLA &#8212; was examined using immunocytochemistry, and labeling with antibodies directed towards CNTFR&#945; and the late-endosome/lysosome marker Lysosomal-associated Membrane Protein 1 (LAMP-1). The experiment was performed on cells treated as well as untreated with inhibitors of lysosomal enzymes. The idea was of course that if CNTFR&#945; was degraded in lysosomes, cells treated with enzyme-inhibitors would present CNTFR&#945;-staining accumulating in LAMP-1 positive vesicles, whereas untreated cells would show little or no staining for CNTFR&#945;.
Does CNTFR&#945; downregulation lower the cellular response to CLC:CLF-1 stimulation? The trimeric complex consisting of CLC, CLF-1, and CNTFR&#945; signals via the gp130/LIFR&#946; heterodimer using the JAK/STAT3 pathway. Accordingly, the capacity of cells to respond to CLC:CLF-1 signaling upon downregulation of CNTFR&#945; was explored by Western blot detection and quantitation of the phospho-STAT3 (pSTAT3)/total STAT3 level in sorLA transfectants.

<table><tbody><tr><td>Zeocin</td><td>InvivoGen</td><td>ant-zn-1</td><td></td></tr><tr><td>Hygromycin B Gold</td><td>InvivoGen</td><td>ant-hg-1</td><td></td></tr><tr><td>FuGENE 6 Transfection Reagent&amp;nbsp;</td><td>Roche</td><td>11 815 091 001</td><td></td></tr><tr><td>4-well plates (Nunclon Delta Surface)</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>Safe-Lock tubes</td><td>Eppendorf</td><td>0030 120.086</td><td></td></tr><tr><td>Dulbecco Modified Eagle&amp;acute;s Medium (DMEM)</td><td>Lonza</td><td>BE12-604F/U1</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Scientific</td><td>10270106</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Scientific</td><td>15140122</td><td></td></tr><tr><td>Dulbecco Phosphate Buffered Saline&amp;nbsp; (DPBS)</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>CLC:CLF-1</td><td>R&amp;amp;D Systems</td><td>1151-CL/CF</td><td></td></tr><tr><td>Mouse anti-LAMP-1&amp;nbsp;</td><td>DSHP</td><td>H4A3</td><td></td></tr><tr><td>Rabbit anti-sorLA</td><td></td><td></td><td>Reference 16</td></tr><tr><td>Mouse anti-b-actin&amp;nbsp;</td><td>Sigma</td><td>A2228</td><td></td></tr><tr><td>Mouse anti-Myc</td><td>Genscript</td><td>A00704</td><td></td></tr><tr><td>Goat anti-CNTFRa&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>AF-303</td><td></td></tr><tr><td>Rabbit anti-pSTAT3&amp;nbsp;</td><td>Cell signaling Technology</td><td>9145s</td><td></td></tr><tr><td>Mouse anti-STAT3&amp;nbsp;</td><td>Cell signaling Technology</td><td>9139s</td><td></td></tr><tr><td>Anti-mouse HRP-linked antibody</td><td>Cell signaling Technology</td><td>7076s</td><td></td></tr><tr><td>Anti-rabbit HRP-linked antibody</td><td>Cell signaling Technology</td><td>7074s</td><td></td></tr><tr><td>Anti-goat HRP-linked antibody</td><td>Dako</td><td>P0160</td><td></td></tr><tr><td>Donkey anti-goat secondary antibody, Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>A11055</td><td></td></tr><tr><td>Donkey anti-mouse secondary antibody, Alexa Fluor 568 conjugate</td><td>Invitrogen</td><td>A10037</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L8511-5MG</td><td></td></tr><tr><td>Pepstatin A&amp;nbsp;</td><td>Sigma</td><td>P4265-5MG</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>AppliChem</td><td>A1388</td><td></td></tr><tr><td>Tris-HCl</td><td>Merck</td><td>1,082,191,000</td><td></td></tr><tr><td>EDTA:Titriplex III</td><td>Merck</td><td>1,084,180,250</td><td></td></tr><tr><td>cOmplete mini EDTA-free</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>04 906 837 001</td><td></td></tr><tr><td>LDS sample Buffer (4x)</td><td>Novex</td><td>NP0007</td><td></td></tr><tr><td>Bio-Rad Protein Assay</td><td>Bio-Rad</td><td>500-0006</td><td></td></tr><tr><td>4% paraformaldehyde (PFA)</td><td>VWR</td><td>97,135,000</td><td></td></tr><tr><td>Saponin</td><td>Sigma</td><td>S7900-100G</td><td></td></tr><tr><td>Mounting Media</td><td>Dako</td><td>S3023</td><td></td></tr></tbody></table>

sorla and clc:clf-1-dependent downregulation of cntfr&#945; as demonstrated by western blotting, inhibition of lysosomal enzymes, and immunocytochemistry

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR&#945; to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-&#946; (gp130/LIFR&#946;) for signaling. Both CLC and CNTFR&#945; are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR&#945; (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFR&#945;) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR&#945; and sorLA, CNTFR&#945; first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR&#945;, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1.
The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFR&#945; expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR&#945;; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR&#945;.

Figure 1 shows a schematic representation of the cell-seeding and CLC:CLF-1 incubation in protocol 1. Figure 2 shows a schematic representation of the cell-seeding and leu/pep and CLC:CLF-1 incubation in protocol 2. Figure 3 shows a schematic representation of the cell-seeding and CLC:CLF-1 incubation and stimulation in protocol 3. Figure 4 shows the sorLA-mediated CLC:CLF-1-dependent downregulation of CNTFR&#945;. Figure 5 shows the sorLA-mediated endocytosis and lysosomal targeting of CNTFR&#945;. Figure 6 shows the lower response to CLC:CLF-1 stimulation after CNTFR&#945; downregulation.
Note that bands signifying CNTFR&#945;-Myc have similar density at time zero, whereas downregulation of CNTFR&#945;-Myc is demonstrated by the weaker band in sorLA transfectants after 5 h (Figure 4). Figure 5 shows that CNTFR&#945;-Myc has accumulated in LAMP-1 positive vesicles of leu/pep treated cells. In contrast, no accumulation of CNTFR&#945;-Myc is seen in cells not subjected to leu/pep treatment. This demonstrates that CNTFR&#945;-Myc is sorted to lysosomes and degraded. As can be seen in Figure 6, the level of pSTAT3 is significantly reduced in pre-stimulated cells as compared to the level in cells that has not been exposed to CLC:CLF-1. This is in accordance with the observed sorLA-mediated downregulation of CNTFR&#945; in the pre-stimulated cells (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/55019/55019fig1.jpg" /> 
Figure 1: Schematic representation of cell-seeding and CLC:CLF-1 incubation in protocol 1. Seed HEK293-CNTFR&#945;-Myc and HEK293-CNTFR&#945;-Myc/sorLA cells in two 4-well plates (0 and 5 h) and incubate cells with CLC:CLF-1 as indicated. <a href="http://cloudflare.jove.com/files/ftp_upload/55019/55019fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55019/55019fig2.jpg" /> 
Figure 2: Schematic representation of cell-seeding and leu/pep and CLC:CLF-1 incubation in protocol 2. Seed HEK293-CNTFR&#945;-Myc/sorLA cells on cover slides in a&#160;4-well plate and incubate cells with or without leu/pep and CLC:CLF-1 as indicated. <a href="http://cloudflare.jove.com/files/ftp_upload/55019/55019fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55019/55019fig3.jpg" /> 
Figure 3: Schematic representation of cell-seeding and CLC:CLF incubation and stimulation in protocol 3. Seed HEK293-sorLA cells in one 4-well plate and incubate cells with or without CLC:CLF-1 (pre-exp.) to downregulate the CNTFR&#945; followed by CLC:CLF-1 stimulation (stim.) to initiate STAT3 phosphorylation as indicated. <a href="http://cloudflare.jove.com/files/ftp_upload/55019/55019fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55019/55019fig4.jpg" /> 
Figure 4: Results showing that SorLA mediates CLC:CLF-1-dependent downregulation of CNTFR&#945;. (A) HEK293-CNTFR&#945;-Myc and (B) HEK293-CNTFR&#945;-Myc/sorLA cells were incubated in the absence (white columns) or presence (grey columns) of 10 nM CLC:CLF-1. After 0 and 5 h, the incubation was stopped and the content of CNTFR&#945;-Myc in the medium (m) and in the cell lysates (l) was detected by Western blotting and quantified by densitometry. The upper panels show Western blot results from representative experiments and the lower panels show the detected levels of CNTFR&#945;-Myc found in the cell lysates. The levels are shown relative to the CNTFR&#945;-Myc level in the single and double transfectants at 0 h. Each column represents mean &#177;SEM (n = 3). p-values calculated using t-test. Reproduced after original figure13.&#160;<a href="http://cloudflare.jove.com/files/ftp_upload/55019/55019fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 5" src="/files/ftp_upload/55019/55019fig5.jpg" /> 
Figure 5: Results showing that SorLA mediates endocytosis and lysosomal targeting of CNTFR&#945;. HEK293-CNTFR&#945;-Myc/sorLA cells were treated with or without leu/pep, incubated with 10 nM CLC:CLF-1 for 5 h, fixed, and finally stained using anti-CNTFR&#945; and anti-LAMP-1 antibodies as described in protocol 2. Scale bars: 5 &#956;m. Reproduced after original figure13. <a href="http://cloudflare.jove.com/files/ftp_upload/55019/55019fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55019/55019fig6.jpg" /> 
Figure 6: SorLA-mediated downregulation of CNTFR&#945; is accompanied by a lowered cellular response to CLC:CLF-1 stimulation. 
HEK293-sorLA cells were pre-incubated in the absence or presence of 10 nM CLC:CLF-1 for 5 h (pre-exp.), starved in blank medium for 90 min, and stimulated with 5 nM CLC:CLF-1 for 15 min (stim.). (left) The columns show the relative levels of pSTAT3 in the cells. Each column represents mean &#177;SEM (n = 3) relative to the pSTAT3 level in cells preincubated in the absence of CLC:CLF-1 but stimulated with CLC:CLF-1. p-value calculated using t-test. (right) The Western blot shows the response of pSTAT3 and STAT3 obtained in a representative experiment. Reproduced after original figure13. <a href="http://cloudflare.jove.com/files/ftp_upload/55019/55019fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about SorLA and CLC:CLF-1-dependent Downregulation of CNTFRÎ± as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry at JoV

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-&#945; (CNTFR&#945;), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored ...

sorla-clcclf-1-dependent-downregulation-cntfr-as-demonstrated-western

Research

JoVE Journal

Biochemistry

7.3K Views.  Aarhus University. The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-α (CNTFRα), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

Video: SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Cancer Research

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Genetics

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Neuroscience

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Biology

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

FRET-Based Method to Monitor LRRC8 Channel Activity Without Electrophysiology

Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity Using Sensitized-Emission F&#246;rster Resonance Energy Transfer (SE-FRET)

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion channels (VRACs)/volume-sensitive outwardly rectifying channels (VSORs), they are activated upon osmotic cell swelling and mediate the extrusion of chloride and organic osmolytes, leading to the efflux of water and hence cell shrinkage. Beyond their role in osmotic volume regulation, VRACs have been implicated in cellular processes such as differentiation, migration, and apoptosis. Through their effect on membrane potential and their transport of various signaling molecules, leucine-rich repeat containing 8 (LRRC8) channels play roles in neuron-glia communication, insulin secretion, and immune response. The activation mechanism has remained elusive. LRRC8 channels, like other ion channels, are typically studied using electrophysiological methods. Here, we describe a method to detect LRRC8 channel activation by measuring intra-complex sensitized-emission F&#246;rster resonance energy transfer (SE-FRET) between fluorescent proteins fused to the C-terminal leucine-rich repeat domains of LRRC8 subunits. This method offers the possibility to study channel activation in situ without exchange of the cytosolic environment and during processes such as cell differentiation and apoptosis.

Electrophysiology is the gold standard for investigating ion channel activity. However, there are plenty of alternative approaches, including optical methods. Here, we describe a method to monitor the activity of the leucine-rich repeat containing 8 channel (LRRC8)-formed anion channels using an inter-subunit F&#246;rster resonance energy transfer (FRET)-based method.

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Immunology and Infection

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis.
There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation.
Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy.
We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress.
This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

In the present study, the expression is knocked down of two downstream signaling components of the PERK pathway, the cytoprotective calcineurin and the pro-apoptotic CHOP, by using specific shRNAs. In opposite ways, these modulate the susceptibility of primary cortical neurons to neurite atrophy after induction of endoplasmic reticulum stress.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) ...

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