title: Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS.
abstract: Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or by pharmacological block of Src family kinase signaling, consistent with receptor recruitment requiring netrin-1-activated signaling. Our findings reveal a mechanism activated by netrin-1 that recruits DCC and UNC5B to the plasma membrane.
authors: Department of Chemistry, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; McGill Program in Neuroengineering, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Angelica A
authors: AA
authors: Gopal
authors: McGill Program in Neuroengineering, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; Department of Physics, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Benjamin
authors: B
authors: Rappaz
authors: Department of Chemistry, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Vincent
authors: V
authors: Rouger
authors: Department of Physics, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Iain B
authors: IB
authors: Martyn
authors: Department of Physics, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Peter D
authors: PD
authors: Dahlberg
authors: Department of Chemistry, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Rachel J
authors: RJ
authors: Meland
authors: McGill Program in Neuroengineering, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Ian V
authors: IV
authors: Beamish
authors: McGill Program in Neuroengineering, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors: Timothy E
authors: TE
authors: Kennedy
authors: Department of Chemistry, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; McGill Program in Neuroengineering, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; Department of Physics, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada. Electronic address: paul.wiseman@mcgill.ca.
authors: Paul W
authors: PW
authors: Wiseman
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D000075389:DCC Receptor; D057809:HEK293 Cells; D006801:Humans; D009414:Nerve Growth Factors / Q000378:metabolism*; D000075386:Netrin Receptors; D000075388:Netrin-1; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D000075389:DCC Receptor; C501909:DCC protein, human; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D000075386:Netrin Receptors; D011956:Receptors, Cell Surface; D025521:Tumor Suppressor Proteins; C532445:UNC5B protein, human; D000075388:Netrin-1