Potential cellular functions of N-ethylmaleimide sensitive factor in the photoreceptor

Abstract

Recent work has established potential new functional roles for NSF in the photoreceptor. First, the interaction of Arr1 and NSF is ATP-dependent, and the N-terminal domain of Arr1 interacts with the N and D1 junctional domains of NSF. The Arr1-NSF interactions are greater in the photoreceptor synaptic terminal in the dark. Furthermore, Arr1 enhances the NSF ATPase activity and increases the NSF disassembly activities, which are critical for NSF functions in sustaining a higher rate of exocytosis in the photoreceptor synapses and the compensatory endocytosis to retrieve vesicle membrane and vesicle proteins for vesicle recycling. These data demonstrate the Arr1 and NSF interaction are necessary for both maintenance and modulation of normal photoreceptor synaptic regulation. Second, NSF colocalizes and specifically binds to RP2, especially in the ciliary and synaptic region of the photoreceptor, and NSF-RP2 interaction may play an important role in membrane protein trafficking in the photoreceptor. Inherited retinal degeneration affects about 1 in 2,000-3,000 individuals in the world and is the leading cause of visual loss in young people and accounts for a large proportion of blindness in adult life. These studies accelerate our ability to gain insight into the diverse roles of the NSF in the photoreceptor cells and enable us to understand more precisely the molecular mechanisms underlying night blindness associated with clinically diagnosed Oguchi disease or other forms of retinitis pigmentosa.

Fig. 101.1

Structure of NSF domains. Each NSF monomer is comprised of three domains: an N-terminal domain (amino acid 1–205) that is responsible for the interaction with a-SNAP and SNAREs; and two homologous ATP-binding domains, D1 (amino acids 206–477), in which the hydrolytic activity is associated with NSF-driven SNARE complex disassembly, and D2 (amino acids 478–744), which is responsible for hexamer formation. Critical residues involved in posttranslational processing include Cys21, 91 and 264, Tyr83, Ser237, and Ser569

Fig. 101.2

NSF and Arr1 localization in dark-adapted retinas. Immunohistochemical fluorescent labeling of retinal sections with dual localization of NSF and Arr1 staining in the WT mice dark-adapted (DA) overnight and killed in the dark. Adult WT mouse retina frozen sections were triple labeled fluorescently with the antimouse Arr1 MAb D9F2 (red, 1:20,000), antirabbit NSF PAb (green, 1:2,500, Abcam), and secondary antibodies conjugated to Alexa Fluor 488 or 568, respectively (1:500, invitrogen), and DAPI for the nuclei (blue, (a, b)). NSF immunoreactive staining pattern of NSF is mainly in the OPL and IPL in dark-adapted retinas (c, d). Arr1 immunoreactive pattern is predominantly in the inner segment, perinuclear area and a fraction in the photoreceptor terminal in DA retinas ((c), higher magnification; large arrows). OS outer segment; IS inner segments; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; GLC ganglion cell layer. Scale bar, 20 μm in upper panels; 10 μm in lower panels

Fig. 101.3

NSF and Arr1 localization in light-adapted retinas. Immunohistochemical fluorescent labeling of NSF and Arr1 in retinal sections from WT mouse exposed to light for 2 h prior to killing (light-adapted [LA]). Adult WT mouse retina frozen sections were triple labeled fluorescently as in Fig. 101.2 ((a, b) DAPI stains nuclei blue). NSF immunoreactive staining pattern of NSF is mainly in the OPL and IPL in light-adapted retinas (c, d). Arr1 immunoreactive pattern is translocated to the outer segment in LA retinas. Arr1 immunoreactivity is extensively dual localized with NSF immunological staining and limited punctate dual staining in the OPL in LA retina ((d) higher magnification; smaller arrows). Abbreviations are listed in Fig. 101.2. Scale bar, 20 μm in upper panels; 10 μm in lower panels