All reported studies on CSPP isoforms and their centrosome- and MT-associated functions mainly examined their role in facilitating mitosis and cytokinesis (Patzke et al., 2005
; Asiedu et al., 2009
). Our present investigation is therefore the first to shed light on a postmitotic (cytoskeleton associated) function of CSPP proteins in both cells and tissues. Four different experimental strategies used to study CSPP proteins in vitro and in vivo link them functionally to the primary cilium: 1) the analysis of subcellular localization of endogenous and ectopic CSPP proteins in hTERT-RPE1 and primary trachea epithelia cells; 2) the analysis of cell type specific expression in human renal and murine renal, retinal, and bronchial tissue sections in conjunction with the tissue-specific mRNA expression reported during mouse embryogenesis; 3) the analysis of the effects of CSPP overexpression and depletion on cilia length and the efficiency of ciliogenesis of hTERT-RPE1 cells, respectively; and 4) the identification and characterization of the interaction and complex formation with known ciliary proteins NPHP8 and NPHP4.
The immunofluorescence staining suggests that CSPP proteins can occur in different protein pools localized to 1) centrosomes, 2) midspindel/midbodies, and 3) the cilia axoneme. Both antibodies used in this study detected endogenous CSPP isoforms at the centrosome, the midbody and along the cilia axoneme. However, not all CSPP proteins stained by the affinity-purified polyclonal CSPP-L–specific antibody also were stained by the mAb that is directed against the common C-terminal domain of both CSPP isoforms (). This discrepancy may be explained by either the lower affinity of the mAb compared with the CSPP-L–specific polyclonal antibody or epitope masking of the C-terminal domain by the formation of protein complexes such as with NPHP8. Alternatively, because we have shown previously that CSPP proteins can be phosphorylated at serine residues (Patzke et al., 2005
), posttranslational modification could mask the epitope detected by the mAb.
Our results show that CSPP proteins not only are expressed in NPHP protein-expressing cell types in renal, retinal, and respiratory tissue sections but also that they interact directly with NPHP8 and indirectly with NPHP4. Furthermore, the interaction of endogenous CSPP proteins with NPHP8 is not only detected in cell lines but also confirmed in bovine retina extracts. Our results suggest that CSPP proteins are required for correct localization of NPHP8 to the tip of the basal body that is forming the transition zone. In hTERT-RPE1 cells the targeted knockdown of CSPP-L alone is sufficient to impair ciliogenesis and to decrease the recruitment of NPHP8 to centrosomes, indicating its sole significance in this process in this cell line (C and A). Our finding that ciliogenesis is not impaired by NPHP8 knockdown indicates that loss of NPHP8 from the transition zone alone is not sufficient to explain the CSPP-L siRNA-mediated ciliogenesis defect. This finding is consistent with the observation of primary cilia in primary mouse embryonic fibroblasts derived from the NPHP8 knockout mouse (Vierkotten et al., 2007
). The observed increase in cilia length might reflect a homeostatic response in which hTERT-RPE1 cells try to compensate for lack of signaling through the cilium as NPHP8 has been shown to be required for Shh signaling. Alternatively, it could be the consequence of imbalanced sorting of cilia length determining proteins (e.g., Rab8; Nachury et al., 2007
) over the transition zone, because the NPHP8 interactor NPHP4 and its interactor NPHP1 also are suggested to regulate transport through the transition zone (Winkelbauer et al., 2005
; Jauregui et al., 2008
). However, NPHP8 seems to be expendable for the localization of NPHP4 and CSPP-L to the primary cilium. Thus, NPHP8 might act antagonistically on CSPP-L, which we found not only to be required for ciliogenesis but also to positively regulate cilia length upon ectopic expression. Interestingly, NPHP8 has been found to interact with RPGR (Khanna et al., 2009
), which in turn is associated with NPHP6 (CEP290; Chang et al., 2006
) that has been shown to regulate ciliogenesis via recruitment of Rab8 (Kim et al., 2008
; Tsang et al., 2008
). Although our study did not unravel the mechanism of CSPP/CSPP-L dependence in ciliogenesis, we identified a second arm of the NPHP network feeding into the control of ciliogenesis or eventually cilia homeostasis.
Depletion of several different proteins localizing to distinct structural moieties of the centrosome have been shown to result in ciliogenesis defects similar to CSPP/CSPP-L depletion reported here. These studies highlight the importance of the general structural integrity of the centrosome (Mikule et al., 2007
; Graser et al., 2007
). However, based on the fact that we found CSPP-L to contain an actin- or stress fiber-targeting domain in its isoform specific N-terminal domain (Supplemental Figure 3), one may speculate that CSPP-L helps to position the centrosome during its maturation/morphogenesis to the basal body in the progress of ciliogenesis. Indeed, actin remodeling is required for ciliogenesis and cilia positioning. Contractile proteins such as myosin are constituents of basal bodies (Gordon et al., 1980
; Gordon and Lane, 1984
; Dawe et al., 2007
) and a role of actin modulation in ciliogenesis, maintenance, and function is starting to emerge (Kim et al., 2010
; Molla-Herman et al., 2010
). Interestingly, CSPP-L has been shown to recruit a myosin GTPase exchange factor MyoGEF to facilitate actomyosin ring contraction during cell division (Asiedu et al., 2009
). Hence, localized organization or activation of the actomyosin network might be the common functional denominator for CSPP-L at the centrosome during ciliogenesis and at the midspindle/midbody during cell division that is also found in nonciliated lymphocytes (Supplemental Figure 2). In this context, it is interesting to note that the C-terminal domain of CSPP and CSPP-L that is required for interaction with NPHP8 was found previously to be required to restrict the MT organizing activity of CSPP proteins to mitosis (Patzke et al., 2006
). NPHP8 could be a modulator of this MT-organizing activity and could thereby contribute to cilia assembly/maintenance.
The interaction of NPHP8 with CSPP proteins still allowed ternary complex formation with NPHP4, which in turn is a known NPHP1 interacting protein (Mollet et al., 2005
). The partial colocalization of CSPP-L and NPHP4 as well as NPHP1 at the basal body/transition zone (E) may be supportive for the existence of CSPP-L–NPHP8–NPHP4 (–NPHP1) complex formation in cells. Furthermore, CSPP proteins are enriched at the connecting cilium and at synapses of rod and cone cells in the outer nuclear layer (), thus closely resembling reported staining pattern of NPHP8 and NPHP4 (see supplemental figure 5 in Arts et al., 2007
). Also, NPHP1 is a known constituent of the retinal connecting cilium as well as of the transition zone of the renal and respiratory cilia (Fliegauf et al., 2006
; Jiang et al., 2009
). Notably, in addition to their ciliary localization, these proteins also have been shown to localize to cell–cell and cell–matrix contacts where they complex with proteins involved in cytoskeleton organization to facilitate epithelial morphogenesis (Donaldson et al., 2000
; Mollet et al., 2005
; Delous et al., 2009
); and importantly, NPHP associated mutations in NPHP8 weaken or abrogate their interaction with NPHP4 (Arts et al., 2007
; Delous et al., 2007
). Although little is known about the dynamic behavior of these proteins, in a unifying concept NPHP8 might modulate cilia growth and polarization on two levels: indirectly through modulation of transport across the transition zone thereby affecting planar-cell-polarity signaling pathways and directly through interactions with cytoskeleton affecting proteins such as NPHP4, NPHP6, and Rab8 via RPGR, and CSPP proteins identified here (Supplemental Figure 7). Our results may suggest that CSPP-L and NPHP8 can form a functional unit of which (in hTERT-RPE1 cells) CSPP-L is required for cilia formation and NPHP8 recruitment or maintenance. Once the cilium is formed, NPHP8 might antagonistically act on CSPP-L to regulate cilia length. This might involve further interactions with other proteins such as NPHP4. However, additional work is required to investigate whether, when, and where these proteins can occur as multiprotein complexes together with CSPP in vivo. Furthermore the identification of putative binding partners of CSPP-L within the ciliary axoneme and its tip region will be a prerequisite to understand its effect on axoneme length control.
Finally, like many NPHP proteins, CSPP is highly conserved in vertebrate evolution (Patzke et al., 2005
). Although unlike NPHP1 and NPHP4, no CSPP homologues were identifiable in genomes of the flagellated chlorophyte Chlamydomonas rheinhardtii
or in genomes of nematodes, we identified a homologue in the choanoflagellate Monosiga brevicollis
(Supplemental Figure 8), one of the closest unicellular relatives of animals. This phylogenetic profile is reminiscent of that of the here identified CSPP-interacting protein NPHP8 (Vierkotten et al., 2007
). The vertebrate-specific coevolution may strengthen the hypothesis of a cell-type–specific functional relation of both proteins.
To conclude, our investigation of CSPP expression at cellular and tissue levels identify a novel, nonmitotic function for CSPP isoforms and place these proteins into a NPHP protein containing network at the primary cilium. The activity of this network is crucial for normal renal and retinal tissue architecture and function. Importantly, the larger isoform CSPP-L is found to be required for cilia formation and found to promote cilia prolongation. To date, 10 disease genes have been identified accounting collectively for only ~30% of the nephronophthisis patients (Hildebrandt et al., 2009a
; Otto et al., 2009
). Our results define CSPP1
as a novel candidate gene for multiorgan phenotype ciliopathies. The putative role of CSPP proteins in human ciliopathies, in particular nephronophthisis-associated ciliopathies, should therefore be investigated.