Journal: bioRxiv

Article Title: CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization

doi: 10.1101/2024.11.06.622207

Figure Lengend Snippet: (A) Schematic of the human CDR2 protein and sequence alignment of its N-terminal CC1 box (motif AAXXG) with that of other human dynein adaptors (see also ). The CC1 box binds DLIC, as illustrated in the cartoon below the alignment. (B) Elution profiles and BlueSafe-stained SDS-PAGE gels of purified recombinant human CDR2 and DLIC1 fragments after SEC. The elution profile and gel for DLIC1 are shown on both left and right to facilitate comparison between wild-type (WT) CDR2 and the ΔCC1 box mutant. Molecular weight is indicated in kilodaltons (kDa). (C) BlueSafe-stained SDS-PAGE gels of purified recombinant proteins prior to addition of glutathione agarose resin (Input) and after elution from the resin (GST pull-down), showing that CDR2L binds to DLIC1. (D) BlueSafe-stained SDS-PAGE gel and immunoblot after pull-down of purified recombinant proteins, C-terminally tagged with StTgII, from porcine brain lysate. In the HBS1_6A construct, 6 residues in CDR2’s predicted dynein heavy chain-binding site (HBS1) are mutated to alanine, as shown in . (E) In vitro motility assays with TMR-labeled dynein, dynactin, Lis1 and adaptor fragments. Representative kymographs and the number of processive events per micrometer of microtubule per minute (mean ± SD of 3-4 technical replicates) are shown. The total number of events analyzed were 21 (DDL), 344 (CDR2L 1-159 ), 278 (CDR2L 1-290 ), 69 (CDR2L 1-290 ΔCC1) and 304 (JIP3 1-185 ). Statistical significance was determined using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. **** P < 0.0001; ns = not significant, P > 0.05.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-T (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 0.2% (v/v) Tween 20) for 1 hour and probed overnight at 4°C with the following primary antibodies diluted in 5% non-fat dry milk/TBS-T: mouse monoclonal anti-p150 clone 1/p150Glued (BD Transduction Laboratories 610473; 1:2500), mouse monoclonal anti-DIC clone 74.1 ( ; 1:5000), mouse monoclonal anti-GAPDH clone 1E6D9 (Proteintech 60004-1-Ig; 1:5000), mouse monoclonal anti-α-tubulin clone B-5-1-2 (Merck T5168; 1:5000), mouse monoclonal anti-EF-18 clone A-5 (Santa Cruz Biotechnology sc-393731; 1:2000), rabbit monoclonal anti-KTN1 clone D5F7J (Cell Signaling Technology #13243; 1:2000), rabbit polyclonal anti-CDR2 (Merck HPA023870; 1:1000), rabbit polyclonal anti-CDR2L (Proteintech 14563-1-AP; 1:2000).

Techniques: Sequencing, Staining, SDS Page, Purification, Recombinant, Comparison, Mutagenesis, Molecular Weight, Western Blot, Construct, Binding Assay, In Vitro, Labeling

Journal: bioRxiv

Article Title: CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization

doi: 10.1101/2024.11.06.622207

Figure Lengend Snippet: (A) AF2 model and PAE plot of the CDR2 N-terminal coiled-coil in complex with the DLIC1 C-terminal helix and an N-terminal DHC fragment, which in turn is bound to the WD40 domain of DIC2. (B) Sequence alignment of the CC1 box and the dynein heavy chain binding site 1 (HBS1) in CDR2 and CDR2L proteins from different species (note invertebrates possess a single CDR2/CDR2L homolog). The HBS1 sequence is divergent from that of other adaptors but the interaction is predicted at the correct distance from the CC1 box. 6 residues, marked with asterisks, were mutated to alanine (HBS1_6A mutant) based on sequence conservation among CDR2 proteins and their position in the predicted structure. Accession numbers: CDR2_HUMAN (UniProt Q01850 ), CDR2L_HUMAN (UniProt Q86X02), CDR2_MOUSE (UniProt P97817 ), CDR2L_MOUSE (UniProt A2A6T1), CDR2_XENTR (UniProt F6R4S1), CDR2L_XENTR (UniProt A0A803JSM3), CDR2_DANRE (UniProt E7FC97), CDR2L_DANRE (UniProt Q6NZT2), CDR2_BRABE (UniProt A0A6P4ZS94), CDR2_SACKO (NCBI Reference Sequence XP_002736317.2), CDR2_STRPU (UniProt A0A7M7NRE1), CDR2_LINAN (NCBI Reference Sequence XP_013392376.1), CEN_DROME (UniProt Q9VIK6), CDR2_HYDVU (UniProt A0A8B6XII3). Species key (Phylum): HUMAN, Homo sapiens (Chordata); MOUSE, Mus musculus (Chordata); XENTR, Xenopus tropicalis (Chordata); DANRE, Danio rerio (Chordata); BRABE, Branchiostoma belcheri (Chordata); SACKO, Saccoglossus kowalevskii (Hemichordata); STRPU, Strongylocentrotus purpuratus (Echinodermata); LINAN, Lingula anatina (Brachiopoda); DROME, Drosophila melanogaster (Arthropoda); HYDVU, Hydra vulgaris (Cnidaria). (C) Elution profiles and BlueSafe-stained SDS-PAGE gels of purified recombinant human CDR2 and DLIC1 fragments after SEC. DLIC1-C corresponds to residues 388-523. The elution profile and gel for CDR2 are shown on both left and right to facilitate comparison between wild-type DLIC1-C and the F447A/F448A mutant. Molecular weight is indicated in kilodaltons (kDa).

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-T (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 0.2% (v/v) Tween 20) for 1 hour and probed overnight at 4°C with the following primary antibodies diluted in 5% non-fat dry milk/TBS-T: mouse monoclonal anti-p150 clone 1/p150Glued (BD Transduction Laboratories 610473; 1:2500), mouse monoclonal anti-DIC clone 74.1 ( ; 1:5000), mouse monoclonal anti-GAPDH clone 1E6D9 (Proteintech 60004-1-Ig; 1:5000), mouse monoclonal anti-α-tubulin clone B-5-1-2 (Merck T5168; 1:5000), mouse monoclonal anti-EF-18 clone A-5 (Santa Cruz Biotechnology sc-393731; 1:2000), rabbit monoclonal anti-KTN1 clone D5F7J (Cell Signaling Technology #13243; 1:2000), rabbit polyclonal anti-CDR2 (Merck HPA023870; 1:1000), rabbit polyclonal anti-CDR2L (Proteintech 14563-1-AP; 1:2000).

Techniques: Sequencing, Binding Assay, Mutagenesis, Staining, SDS Page, Purification, Recombinant, Comparison, Molecular Weight

Journal: bioRxiv

Article Title: CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization

doi: 10.1101/2024.11.06.622207

Figure Lengend Snippet: (A) Immunoblots of HeLa cells harboring single and double KOs of CDR2 and CDR2L (two independently derived cell lines were analyzed for each condition). GAPDH serves as the loading control. Molecular weight is indicated in kilodaltons (kDa). (B) Immunoblots of CDR2/L double KO cells stably expressing GFP::3xFLAG::CDR2 or CDR2L, used for the experiments in . GAPDH serves as the loading control. Molecular weight is indicated in kilodaltons (kDa). (C) Immunofluorescence of CDR2/L double KO cells stably expressing GFP::3xFLAG::CDR2L, showing co-localization with KTN1 and diffuse cytoplasmic signal. Note that while average expression levels of transgene-encoded CDR2L are significantly higher than those of endogenous CDR2L, as shown in (B) , expression in individual cells is variable. Cells shown here have relatively low expression levels. Scale bar, 10 µm. (D) Sequence alignment of the C-terminal helix in CDR2 and CDR2L proteins from different species. Accession numbers and species key as in . (E) –(G) Immunofluorescence images and immunoblots showing knockdown of KTN1 by RNAi and the resulting delocalization/destabilization of CDR2 in HeLa cells. By contrast, KTN1 levels remain unaffected in CDR2/L double KO cells (two independently derived KO cell lines were analyzed). Scale bars, 20 µm (E) and 10 µm (F) . Molecular weight is indicated in kilodaltons (kDa). (H) Sequence alignment of the CDR2/eEF1Bβ binding site in KTN1 and its paralog RRBP1 (p180) from different species (invertebrates possess a single KTN1/RRBP1 homolog). Accession numbers: KTN1_HUMAN (UniProt Q86UP2), RRBP1_HUMAN (Q9P2E9), KTN1_MOUSE (UniProt Q61595 ), RRBP1_MOUSE (UniProt Q99PL5), KTN1_XENTR (UniProt B3DL66), RRBP1_XENTR (UniProt F7A6K6), KTN1_DANRE (UniProt E7F049), RRBP1_DANRE (UniProt B8A4D7), RRBP1_BRABE (UniProt A0A6P5A3T7), RRBP1_SACKO (NCBI Reference Sequence XP_002741373.1), RRBP1_STRPU (A0A7M7LVI4), KTN1_LINAN (NCBI Reference Sequence XP_013397491.1). Species key as in . No CDR2 binding site could be identified for the KTN1/RRBP1 homologs of DROME and HYDVU (UniProt Q960Y8 and T2M451, respectively), despite the presence of a well conserved CDR2 helix, as shown in (D) .

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-T (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 0.2% (v/v) Tween 20) for 1 hour and probed overnight at 4°C with the following primary antibodies diluted in 5% non-fat dry milk/TBS-T: mouse monoclonal anti-p150 clone 1/p150Glued (BD Transduction Laboratories 610473; 1:2500), mouse monoclonal anti-DIC clone 74.1 ( ; 1:5000), mouse monoclonal anti-GAPDH clone 1E6D9 (Proteintech 60004-1-Ig; 1:5000), mouse monoclonal anti-α-tubulin clone B-5-1-2 (Merck T5168; 1:5000), mouse monoclonal anti-EF-18 clone A-5 (Santa Cruz Biotechnology sc-393731; 1:2000), rabbit monoclonal anti-KTN1 clone D5F7J (Cell Signaling Technology #13243; 1:2000), rabbit polyclonal anti-CDR2 (Merck HPA023870; 1:1000), rabbit polyclonal anti-CDR2L (Proteintech 14563-1-AP; 1:2000).

Techniques: Western Blot, Derivative Assay, Control, Molecular Weight, Stable Transfection, Expressing, Immunofluorescence, Sequencing, Knockdown, Binding Assay

Journal: bioRxiv

Article Title: CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization

doi: 10.1101/2024.11.06.622207

Figure Lengend Snippet: (A) Schematic illustrating construction of HeLa CDR2/L double KO cell lines stably expressing exogenous GFP::3xFLAG-tagged CDR2 or CDR2L used for immunoprecipitation followed by quantitative mass spectrometry. The relative abundance of KTN1 and RRBP1/p180 in anti-FLAG immunoprecipitations from transgenic and parental CDR2/L double KO cells is shown for two independent experiments (Exp1 and 2) on the right. (B) Immunofluorescence image of a HeLa cell stably expressing GFP::3xFLAG-tagged CDR2, showing co-localization with the ER sheet protein KTN1. Scale bar, 5 µm. (C) Immunofluorescence showing co-localization of endogenous CDR2 with the ER sheet protein CLIMP63. A CDR2 KO cell serves as the control for CDR2 antibody specificity. Scale bar, 5 µm. (D) AF2 model and predicted alignment error (PAE) plot of the KTN1 C-terminal coiled-coil domain in complex with the C-terminal helix of CDR2. KTN1 domain organization and C-terminal KTN1 fragments (KTN1-C) used for in vitro binding assays in (E) are also shown. (E) Elution profiles and BlueSafe-stained SDS-PAGE gels of purified recombinant human CDR2 and KTN1 fragments after SEC. The elution profile and gel for CDR2 are shown on both left and right to facilitate comparison between wild-type KTN1-C and the Δ1114–1153 mutant. Molecular weight is indicated in kilodaltons (kDa). (F) Immunofluorescence images of HeLa CDR2/L double KO cells transiently expressing GFP::CDR2 with and without its C-terminal helix, demonstrating that the helix is necessary and sufficient for ER localization. Scale bar, 5 µm. (G) Cartoon of the dynein recruitment pathway at ER sheets, based on results from in vitro reconstitution of protein–protein interactions and cell-based assays with binding-deficient mutants.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-T (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 0.2% (v/v) Tween 20) for 1 hour and probed overnight at 4°C with the following primary antibodies diluted in 5% non-fat dry milk/TBS-T: mouse monoclonal anti-p150 clone 1/p150Glued (BD Transduction Laboratories 610473; 1:2500), mouse monoclonal anti-DIC clone 74.1 ( ; 1:5000), mouse monoclonal anti-GAPDH clone 1E6D9 (Proteintech 60004-1-Ig; 1:5000), mouse monoclonal anti-α-tubulin clone B-5-1-2 (Merck T5168; 1:5000), mouse monoclonal anti-EF-18 clone A-5 (Santa Cruz Biotechnology sc-393731; 1:2000), rabbit monoclonal anti-KTN1 clone D5F7J (Cell Signaling Technology #13243; 1:2000), rabbit polyclonal anti-CDR2 (Merck HPA023870; 1:1000), rabbit polyclonal anti-CDR2L (Proteintech 14563-1-AP; 1:2000).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Mass Spectrometry, Transgenic Assay, Immunofluorescence, Control, In Vitro, Binding Assay, Staining, SDS Page, Purification, Recombinant, Comparison, Mutagenesis, Molecular Weight

Journal: Annals of Clinical and Translational Neurology

Article Title: Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

doi: 10.1002/acn3.51212

Figure Lengend Snippet: Antibody specificities determined by mass spectrometry analysis of CDR2L and CDR2 proteins immunoprecipitated from OvCar3 and HepG2 cell lysates.

Article Snippet: Primary antibodies against Hsp60 (EnCor Biotechnology, #CPCA‐HSP60), CDR2 (Sigma‐Aldrich, #018151), CDR2L (Proteintech, #14563‐1‐AP), SON, and SRSF2 were applied for 1 h (1:100 in blocking solution), followed by 3x 5‐minute washes with Wash Buffer A supplied with the kit.

Techniques: Mass Spectrometry, Immunoprecipitation

Journal: Annals of Clinical and Translational Neurology

Article Title: Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

doi: 10.1002/acn3.51212

Figure Lengend Snippet: Protein‐protein interaction networks visualized by STRING. (A) CDR2L was predicted to interact with ribosomal proteins (rpS6, red box). The nodes indicate proteins, and the edges represent protein‐protein associations. (B) Protein‐protein interaction network of nuclear speckles proteins, SON, eIF4A3, and SRSF2, predicted to interact with CDR2. eIF4A3 (red) directly interacts with SON (light green) and SRSF2 (blue). (C) eIF4A3 (yellow) interacts with rpS6 (blue), indicated by colored edges. Predicted binding partners, CDR2L (green) and CDR2 (red), are manually gated (black, dotted lines). Color‐coded edges; light blue: curated databases, dark blue: gene co‐occurrence, pink: experimentally determined, green: text mining. Interactions with a medium score of 0.400 or more are shown.

Article Snippet: Primary antibodies against Hsp60 (EnCor Biotechnology, #CPCA‐HSP60), CDR2 (Sigma‐Aldrich, #018151), CDR2L (Proteintech, #14563‐1‐AP), SON, and SRSF2 were applied for 1 h (1:100 in blocking solution), followed by 3x 5‐minute washes with Wash Buffer A supplied with the kit.

Techniques: Binding Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

doi: 10.1002/acn3.51212

Figure Lengend Snippet: CDR2L co‐localizes with ribosomes and CDR2 with nuclear speckles in OvCar3 cells as shown using proximity ligation assay. (A) Upper row: Co‐localization of anti‐CDR2L (green) and ribosomes (rpS6; red) in the cytoplasm (yellow; merged image). Lower row: Co‐localization of anti‐CDR2 (green) and nuclear speckles (SRSF2; red) in the nucleus (yellow; merged image). (B) Upper row: Positive Duolink (green) between CDR2L and ribosomes (rpS6) in the cytoplasm (hsp60 in magenta was used to show the extent of the cell cytoplasm; merged image). Lower row: Positive Duolink (green) between CDR2 and nuclear speckle marker (SRSF2) in the nuclei; no co‐localization was observed with cytoplasmic marker hsp60 (magenta; merged image). DAPI was used as a marker for the nuclei (blue). Scare bars = 10 µm.

Article Snippet: Primary antibodies against Hsp60 (EnCor Biotechnology, #CPCA‐HSP60), CDR2 (Sigma‐Aldrich, #018151), CDR2L (Proteintech, #14563‐1‐AP), SON, and SRSF2 were applied for 1 h (1:100 in blocking solution), followed by 3x 5‐minute washes with Wash Buffer A supplied with the kit.

Techniques: Proximity Ligation Assay, Marker

Journal: Annals of Clinical and Translational Neurology

Article Title: Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

doi: 10.1002/acn3.51212

Figure Lengend Snippet: CDR2L co‐immunoprecipitates with ribosomal protein rpS6, whereas CDR2 co‐immunoprecipitates with nuclear speckle proteins SON and eIF4A3 in cancer cell lysates. (A) Immunoblot demonstrating the co‐immunoprecipitation of CDR2L and rpS6 from OvCar3 cell lysates. (B) Immunoblot demonstrating the co‐immunoprecipitation of CDR2, SON, and eIF4A3 from HepG2 cell lysates. Input = cancer cell lysates (OvCar3 or HepG2). Beads + lysate = samples that were not treated with primary antibody, and served as negative controls.

Article Snippet: Primary antibodies against Hsp60 (EnCor Biotechnology, #CPCA‐HSP60), CDR2 (Sigma‐Aldrich, #018151), CDR2L (Proteintech, #14563‐1‐AP), SON, and SRSF2 were applied for 1 h (1:100 in blocking solution), followed by 3x 5‐minute washes with Wash Buffer A supplied with the kit.

Techniques: Western Blot, Immunoprecipitation

Journal: Annals of Clinical and Translational Neurology

Article Title: Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

doi: 10.1002/acn3.51212

Figure Lengend Snippet: CDR2L and Yo co‐localize with ribosomal proteins and CDR2 co‐localizes with nuclear speckle proteins in cerebellar Purkinje neurons as shown by super‐resolution microscopy. (A) Upper row: Human cerebellar section stained with Yo‐CSF (green) and anti‐rpS6 (red); the proteins co‐localize in the cytoplasm (yellow; merged image). Middle row: Human cerebellar section stained with anti‐CDR2L (green) and ribosomal marker anti‐rpS6 (red); the proteins co‐localize in the cytoplasm (yellow; merged image). Lower row: Human cerebellar section stained with anti‐CDR2 (green) and nuclear speckle marker anti‐SRSF2 (red); the proteins co‐localize in the nucleus. No co‐localization was found with anti‐Yo (magenta; merged image). (B) Upper row: Rat Purkinje neuron cultures stained with anti‐Yo (CSF; green) and rpS6 (ribosomes; red); co‐localization was observed in the cytoplasm (yellow; merged image). Middle row: Rat Purkinje neuron cultures stained with anti‐CDR2L (green) and anti‐rpS6 (red); co‐localization was observed in the cytoplasm (yellow; merge image). Lower row: Rat Purkinje neurons stained with anti‐CDR2 (green), nuclear speckle protein (red), and anti‐Yo (magenta). CDR2 and the nuclear speckle protein co‐localize in the cell nucleus (yellow; merged image), whereas Yo does not. Scale bars = 10 µm.

Article Snippet: Primary antibodies against Hsp60 (EnCor Biotechnology, #CPCA‐HSP60), CDR2 (Sigma‐Aldrich, #018151), CDR2L (Proteintech, #14563‐1‐AP), SON, and SRSF2 were applied for 1 h (1:100 in blocking solution), followed by 3x 5‐minute washes with Wash Buffer A supplied with the kit.

Techniques: Microscopy, Staining, Marker

Journal: Annals of Clinical and Translational Neurology

Article Title: Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration

doi: 10.1002/acn3.51212

Figure Lengend Snippet: Hypothesis of CDR2L and CDR2 involvement in protein synthesis in Purkinje neurons. CDR2 localizes to the nucleus and directly interacts with nuclear speckle protein eIF4A3. eIF4A3, in conjugation with other cytoplasmic initiation factors, facilitates mRNA binding to the 40S ribosomal subunit. This event is important for mRNA maturation and translation, ultimately resulting in the synthesis of new proteins. CDR2L interacts with ribosomal subunit protein rpS6; therefore, we propose that CDR2L and CDR2 are both involved in the process of protein synthesis. Furthermore, Yo antibody (green) binding to CDR2L in Purkinje neurons of PCD patients may, therefore, interfere with the function of the ribosomal machinery, resulting in disrupted mRNA translation and/or protein synthesis.

Article Snippet: Primary antibodies against Hsp60 (EnCor Biotechnology, #CPCA‐HSP60), CDR2 (Sigma‐Aldrich, #018151), CDR2L (Proteintech, #14563‐1‐AP), SON, and SRSF2 were applied for 1 h (1:100 in blocking solution), followed by 3x 5‐minute washes with Wash Buffer A supplied with the kit.

Techniques: Conjugation Assay, Binding Assay