NAGK-dynein-Golgi interaction at Golgi outpost

NAGK-dynein-Golgi interaction at Golgi outpost

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  Conclusions: Our findings shedlight on the interactions of NAGK with dynein and Golgi outposts in dendritic branch points and their roles in dendritic growth. Further investigation is needed into the molecular mechanisms underlying these interactions. Cultured rat hippocampal neurons were treated with dithiobis (succinimidyl) propionate (DSP+), fixed at DIV10 (stage IV) and double-stained with primary antibodies against N- acetylglucosamine kinase and dynein light-chain roadblock type 1 (a) or dynein heavy chain (b) or kinesin 5B (c). The boxed areas of the merged images are enlarged at the bottom. Colocalized immunopuncta are indicated by arrowheads. Scale bar: 10 μm. Statistics (d). The numbers of puncta per dendritic segment (20 μm, n = 20) were counted and expressed as a percentage of the total (mean±s.d.).**p<0.01. (a) N-acetylglucosamine kinase- interacting proteins identified by using the small domain as bait in yeast two-hybrid screening. (b) The interacting domains of dynein light-chain roadblock type 1. The coding region (amino acids 1–96) of dynein light-chain roadblock type 1 is shown as a bar diagram with the conserved roadblock/LC7 domain. The two positive clones in the yeast two-hybrid selection having a coding region from 3 or 59 to the C-terminal end are also shown. Full length dynein light-chain roadblock type 1 (1-end) and kinesin light chain 1 were used as a positive and a negative control, respectively. The small domain of NAGK interacts with DYNLRB1 in yeast two-hybrid screen NAGK-dynein interactions occur at dendritic branch points NAGK interacts with DYNLRB1 in primary hippocampal neurons Verification of NAGK-dynein complex interaction by proximity ligation assay NAGK-dynein complexes colocalize with Golgi ‘outposts’ at dendritic branch points N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points Md Ariful Islam1 , Syeda Ridita Sharif1 , HyunSook Lee2 , Dae-Hyun Seog3 and Il Soo Moon1,2 1 Department of Anatomy, Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea; 2 Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju, Republic of Korea and 3 Departments of Biochemistry, College of Medicine Inje University, Busan, Republic of Korea N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with ICC for Golgi showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74–96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching. Abstract Co-localization of NAGK-Golgi complexes with DYNLRB1 on MT at dendritic branch points. Ectopic inclusion of small peptides from DYNLRB1 resulted stunted dendrites (b) HEK293T cells were fixed, and PLA was performed using anti-NAGK and anti-dynein light-chain 1 antibodies (NAGK+DYNLL1, upper panel). Alternatively, the cells were transfected with Myc-DDK-tagged NAGK plasmids, and PLA was conducted by using anti-DDK and anti- DYNLL1 antibodies to show the interaction between exogenous NAGK and dynein (DDK+DYNLL1, lower panel). To better depict proximity ligation assay puncta, phase-contrast images were inverted using Photoshop software. Scale bar: 10 μm. (a) Rat hippocampal neurons (DIV2) were fixed, and proximity ligation assay was performed using mouse anti- N-acetylglucosamine kinase and rabbit anti-dynein light- chain roadblock type 1 or anti-dynein heavy chain antibodies. The PLA was followed by immunocytochemistry using a mouse anti-tubulin antibody (green). Boxed areas are enlarged (insets) to show the colocalization of the NAGK-dynein complex with thinner microtubule fibers (arrowheads). Scale bar: 10 μm. (a) N-acetylglucosamine kinase-dynein heavy chain proximity ligation assay was performed in hippocampal neurons (stage IV, DIV 2) and followed by tubulin immunocytochemistry. Proximity ligation assay/phase- contrast (phase) and proximity ligation assay/ immunocytochemistry merged images are shown. The PLA puncta (red) are indicated by arrowheads and with numbers. Scale bar: 10 μm. (b) PLA puncta were sorted into two categories: one with ‘established’ branch joints where the microtubule fiber is already developed (upper panel) and the other at branch initiation sites where the microtubule is yet to develop (lower panel). Positions of branches are indicated with arrowheads. (c) Statistics. Percentages of NAGK-DHC proximity ligation assay puncta at branch joints with or without microtubule in the neonate protrusions are shown by a pie chart. n = 30, 30 neurons. N-acetylglucosamine kinase-dynein heavy chain proximity ligation assay was performed in hippocampal neurons (developmental stage IV) and was followed by immunocytochemistry with an anti-TGN38 antibody to mark Golgi particles. Merged images of proximity ligation assay/ phase-contrast and proximity ligation assay/ immunocytochemistry are shown. (a) PLA signals at soma (1, arrows) and distal branch points (2, 3, arrowheads) are enlarged to show the colocalization of NAGK-dynein complex (red) with Golgi particles (green) oriented to the soma-dendrite joint (inset 1, arrows) or with the Golgi outpost (green) at distal branching sites (inset 2, 3, arrowheads). (b) A high frequency of PLA puncta (red) and Golgi outposts (green) was found at dendritic distensions. Small branches protruding out of the distensions are marked with arrows. Dendritic distensions (boxed areas 1, 2) are enlarged (insets) to show the colocalization of NAGK-dynein complex (red) with Golgi particles (green) marked by arrowheads. Scale bar: 10 μm. (a) NAGK-TGN38 proximity ligation assay (red dots) was followed by ICC with an anti-tubulin antibody (green). The positions of proximity ligation assay signals at branch points are marked with numbers and enlarged in insets. Scale bar: 10 μm. (b) NAGK-GM130 PLA was followed by ICC with an anti-DYNLRB1 antibody. PLA puncta in the soma (box i) were enlarged to show the PLA signals facing the apical dendrite at the somal Golgi apparatus merged with DYNLRB1 signals. PLA dot positions (red) in dendrites are enlarged (box ii, 1–3). Scale bar: 10 μm. (c) Statistics. Pie charts show that ~99% (n = 30 neurons) of NAGK-TGN38 PLA dots were localized to microtubules and ~ 96% (n = 30 neurons) of NAGK-GM130 PLA signals were colocalized with DYNLRB1. Two small peptides are made from DYNLRB1 and named as ‘DYNLRB1 (59-76)’ and ‘DYNLRB1 (74-96)’ . (a) Co-transfection of either ‘DYNLRB1 (59-76)’ or ‘DYNLRB1 (74-96)’ peptide with β-galactosidase was performed and was stained with β-gal staining kit. Neurons transfected with ‘DYNLRB1 (59-76)’ peptide showed healthy dendrites. In contrast, neurons transfected with ‘DYNLRB1 (74-96)’ peptide showed short, stunted dendrites (arrows), while the axon (arrowhead) was apparently unaffected. Scale bar: 10 μm. (b) Sholl analysis. The numbers of dendritic intersections were counted at incremental distances from soma centroids of neurons (n = 30 neurons) transfected with β-gal only (control), with ‘DYNLRB1 (59-76)’ or with ‘DYNLRB1 (74-96)’ . **p<0.01. Results