Abstract
Syne-2 (also known as Nesprin-2) is a member of a family of proteins that are found primarily in the outer nuclear membrane, as well as other subcellular compartments. Syne-2 contains a C-terminal KASH transmembrane domain and is part of a protein network that associates the nuclear envelope to the cytoskeleton via the binding to actin filaments. Syne-2 plays a role in nuclear migration, nuclear positioning during retinal development, and in ciliogenesis. In a previous study, we showed a connection between Syne-2 and the multifunctional scaffold protein Pericentrin (Pcnt). The elimination of the interaction of Syne-2 and Pcnt showed defects in nuclear migration and the formation of outer segments during retinal development, as well as disturbances in centrosomal migration at the beginning of ciliogenesis in general. In this study, the Syne-2 KO mouse model Nesprin-2△ABD (Syne-2tm1Ngl, MGI) with special attention to Pcnt and ciliogenesis was analyzed. We show reduced expression of Syne-2 in the retina of the Syne-2 KO mouse but found no significant structural—and only a minor functional—phenotype. For the first time, detailed expression analyses showed an expression of a Syne-2 protein larger than 400 kDa (~750 kDa) in the Syne-2/Nesprin-2 KO mouse. In conclusion, the lack of an overt phenotype in Syne-2/Nesprin-2 KO mice suggests the usage of alternative translational start sites, producing Syne-2 splice variants with an intact Pcnt interaction site. Nevertheless, deletion of the actin-binding site in the Syne-2/Nesprin-2 KO mouse revealed a high variability in scotopic oscillatory potentials assuming a novel function of Syne-2 in synchronizing inner retinal processes.
Highlights
Syne-2 (Nesprin-2, NM_001005510.2; ENSMUST00000044217) is a nuclear envelope protein that plays a role in nuclear migration, nuclear positioning during retinal development [1,2,3], and in ciliogenesis [4]
Because of the existence of several Syne-2 splice variants, we used the monoclonal antibodies K56-386 [12] and F11 (Santa Cruz), which recognize epitopes mapping near the N- and C-terminus of Syne-2 (Figure 2). Both antibodies detected Syne-2 in the Syne-2/Nesprin-2 KO and WT control retinae with Syne-2 immunoreactivity being present throughout the different retinal layers: the photoreceptor inner segments (IS), the outer nuclear membrane of retinal cells in the outer nuclear layer (ONL), the membrane of the inner nuclear (INL), and the ganglion cell layer (GCL)
Double labeling of Syne-2 and Pcnt showed i) a co-distribution of Syne-2/Pcnt at the ciliary region of the photoreceptors in both genotypes and ii) a Pcnt staining at the centrosomes in the INL and GCL in Nesprin-24ABD KO retinae that did not differ from WT control retinae (Figure 1A’,B’,C’,D’)
Summary
Syne-2 (Nesprin-2, NM_001005510.2; ENSMUST00000044217) is a nuclear envelope protein that plays a role in nuclear migration, nuclear positioning during retinal development [1,2,3], and in ciliogenesis [4]. We provided evidence that the interaction of Pcnt with Syne-2/Nesprin-2 is required for ciliogenesis in differentiated cells, as well as for the attachment of the centrosome to the nucleus in cycling cells, which is crucial for correct nuclear migration and cell polarization. In an effort to achieve a better functional characterization of the Syne-2/Nesprin-2 interaction in the retina, we analyzed in the present study the only available Syne-2 KO mouse model Nesprin-24ABD (Syne-2tm1Ngl , MGI). This mouse lacks the actin-binding domain (ABD) at the N-terminus of Syne-2
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