© 2014. Published by The Company of Biologists Ltd.

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Nephrocystin-4 controls ciliary trafficking of membrane and large soluble proteins at the

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transition zone

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Junya Awata1, Saeko Takada1,4, Clive Standley3, Karl F. Lechtreck1,5, Karl D. Bellvé3, Gregory

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J. Pazour2, Kevin E. Fogarty3 and George B. Witman1

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Department of Cell and Developmental Biology, Program in Molecular Medicine, and

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Biomedical Imaging Group, University of Massachusetts Medical School, Worcester MA 01605

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Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,

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Minneapolis, MN 55455 5

Department of Cellular Biology, University of Georgia, Athens, GA 30602

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Correspondence to:

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George B. Witman

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Department of Cell and Developmental Biology

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University of Massachusetts Medical School

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55 Lake Avenue North

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Worcester, MA 01655

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TEL: 508-856-4038

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FAX: 508-856-1033

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E-mail: [email protected]

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1 JCS Advance Online Article. Posted on 22 August 2014

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ABSTRACT

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The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4

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cause nephronophthisis and blindness in humans. To learn more about NPHP4’s function, we

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have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part

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of the flagellar transition zone, close to the membrane and distal to CEP290, another transition

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zone protein. Therefore, these two proteins, which are incorporated into the transition zone

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independently of each other, define different domains of the transition zone. A nphp4 null

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mutant forms flagella with nearly normal length, ultrastructure, and intraflagellar transport.

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When fractions from isolated wild-type and nphp4 flagella were compared, few differences were

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observed between the axonemes, but a subset of membrane proteins was greatly reduced in the

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mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from

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mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a

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barrier that regulates both membrane and soluble protein composition of flagella. The

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phenotypic consequences of NPHP4 mutations in humans likely follow from protein

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mislocalization due to defects in the TZ barrier.

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INTRODUCTION

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Nephronophthisis (NPHP) is a recessive form of cystic kidney disease that is the most frequent

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genetic cause of chronic renal failure in children (Wolf and Hildebrandt, 2011). To date, 18

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causative human genes for NPHP have been reported (Wolf and Hildebrandt, 2011; Failler et al.,

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2014; Renkema et al., 2014); one of these is NPHP4, which encodes nephrocystin-4 (NPHP4).

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Mutations in NPHP4 lead to juvenile NPHP type 4 as well as retinitis pigmentosa; both disease

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features occur together in Senior-Loken syndrome (Hoefele et al., 2005; Mollet et al., 2002; Otto

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et al., 2002; Schuermann et al., 2002).

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NPHP and Senior-Loken syndrome are ciliopathies (diseases caused by defects in cilia),

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and there is abundant evidence from model systems that loss of NPHP4 causes a ciliary

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phenotype. A study of Nphp4 mutant mice showed that Nphp4 is necessary for normal

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photoreceptor formation and maintenance as well as sperm development (Won et al., 2011). In

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zebrafish, nphp4 morphants exhibited classic ciliary phenotypes, including abnormal body

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curvature and pronephric cysts (Burcklé et al., 2011; Slanchev et al., 2011). In Caenorhabditis

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elegans, RNAi knockdown of NPHP-4 caused defects in male mating behavior, which is

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mediated by sensory cilia (Wolf et al., 2005), and a mutant null for NPHP-4 had mild defects in

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cilia-dependent sensory functions (Winkelbauer et al., 2005). Cilia of the same mutant were

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stunted, misshapen, and had axonemal ultrastructural defects (Jauregui et al., 2008).

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These phenotypes most likely result from defective function of NPHP4 at the ciliary

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transition zone (TZ), a specialized region between the basal body and the cilium proper (Shiba

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and Yokoyama, 2012; Czarnecki and Shah, 2012; Reiter et al., 2012). Several studies have

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reported that NPHP4 localizes to the TZ. LAP-tagged NPHP4 in IMCD3 cells is present at sites

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of cell-cell contact and at the base of the primary cilium, specifically in the TZ (Sang et al.,

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2011). Others have reported NPHP4 in the TZ of cultured mouse renal epithelial cells (Shiba et

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al., 2010) and in the connecting cilium -- a greatly elongated TZ -- of mouse photoreceptor cells

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(Roepman et al., 2005; Patil et al., 2012; Won et al., 2011). In Chlamydomonas reinhardtii,

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NPHP4 was identified in a proteomic study of basal bodies, which included the TZ (Keller et al.,

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2005). In C. elegans, NPHP4 has been localized to the TZ of the sensory cilia (Winkelbauer et

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al., 2005; Jauregui et al., 2008; Williams et al., 2011).

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Evidence is accumulating that the TZ functions as the “pore” or gate proposed to exist at

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the base of the cilium to control access to the ciliary compartment (Rosenbaum and Witman,

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2002). A highly conserved feature of the TZ is the presence of Y-shaped links (Y-links) that

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connect the TZ doublet microtubules to the overlying membrane (Fisch and Dupuis-Williams,

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2011). In C. reinhardtii, these links are partially disrupted in the absence of CEP290, another TZ

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protein, resulting in abnormal flagellar protein composition (Craige et al., 2010). The mutant

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also has defects in intraflagellar transport (IFT), which is used by nearly all organisms to build

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cilia and flagella (Pedersen and Rosenbaum, 2008; Ishikawa and Marshall, 2011). These results

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provided experimental evidence that the TZ functions to control protein and IFT particle entry

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into the cilium, and indicated that CEP290 is important for this function. NPHP4 also may be

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involved in this activity: Cilia of the C. elegans nphp-4 mutant accumulate the membrane-

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associated proteins RGI-2 and TRAM-1a, which normally are excluded from the cilium

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(Williams et al., 2011).

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Despite this progress, we still do not know NPHP4’s precise location in the TZ nor how

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its loss affects overall ciliary composition. In this study, we have used C. reinhardtii to learn

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more about NPHP4. C. reinhardtii has many advantages for studying cilia and ciliary

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components. Particularly relevant for this study, its flagella can be isolated to determine the

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biochemical consequences of loss of NPHP4. NPHP4 is highly conserved (C. reinhardtii to

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human BLASTN E value = 1e-74), so conclusions from studying C. reinhardtii NPHP4 are

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likely to be applicable to humans and other organisms. We found that NPHP4 is located at the

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periphery of the distal TZ, close to the membrane and distal to CEP290. In contrast to CEP290,

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NPHP4 at the TZ does not undergo rapid turnover. We identified an NPHP4 null mutant; the

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mutant has full-length flagella and generally has normal TZ and axonemal ultrastructure. We

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isolated the nphp4 mutant flagella and found that a subset of membrane-associated proteins,

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present in wild-type flagella, are greatly decreased in amount; conversely, the flagella contain

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many large cytosolic housekeeping proteins that normally are excluded from wild-type flagella.

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The results indicate that NPHP4 is a critical component of the selective gate that functions at the

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TZ to control movement of both soluble and membrane-associated proteins between the flagellar

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and cytoplasmic compartments. It is likely that the various phenotypic consequences of NPHP4

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mutations in humans and other organisms all follow from protein mislocalization due to defects

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in the TZ barrier.

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RESULTS

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NPHP4 loss has minor effects on cell motility but slows flagellar assembly

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To identify a C. reinhardtii nphp4 mutant, genomic DNA from our collection of insertional

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mutants (Pazour et al., 1995, 1998) was screened by real-time PCR with primer pairs specific to

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NPHP4. In one strain (B1179), no product was amplified. Further analysis by PCR revealed

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that the mutation, termed nphp4-1, deleted about ~40 kbp around NPHP4 including the entire

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NPHP4 gene, several predicted genes that have no known association with the flagellum, and a

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part of DRC3 (Fig. 1A). DRC3 was first identified in our flagellar proteomic study as FAP134

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(Pazour et al., 2005) and later shown to be a component of the nexin-dynein regulatory complex

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(Lin et al., 2011). To ensure that the phenotype being analyzed in the studies that follow was not

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compromised by the absence of DRC3, B1179 was backcrossed twice to a wild-type strain and

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some of the mutant progeny were then transformed with a DNA fragment containing the DRC3

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gene (Fig. 1A). One of the resulting transformants, rescued for DRC3, was used in this study as

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the nphp4 strain.

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The nphp4 mutant cells are motile and can phototax (J.A., unpublished observations), but

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their swimming paths are slightly more erratic than those of wild-type cells (Fig. 1B). To assess

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this objectively, motility was analyzed using computer-aided sperm analysis (CASA) (Fig. 1C)

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(Mortimer, 2000). Swimming speeds of wild-type and the nphp4 mutant were similar (VCL,

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supplementary material Fig. S1B), but linearity was much lower for the mutant (Fig. 1C; see

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supplementary material Fig. S1A for definition of terms). Transformation of the nphp4 mutant

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with the wild-type NPHP4 gene to generate strain NPHP4-R almost completely restored linearity

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(Fig. 1C), confirming that the phenotype is due to the absence of NPHP4. Consistent with the

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ability to swim nearly normally, steady-state flagella of nphp4 mutant cells are of near normal

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length and appear to be ultrastructurally normal (Fig. 1D, 2A and J.A., unpublished observations).

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Following flagellar amputation, the nphp4 mutant regenerated new flagella more slowly than did

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wild type; normal flagellar regeneration was completely restored in the NPHP4-R strain (Fig.

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1D). Therefore, NPHP4 is needed to build the flagella with normal kinetics.

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NPHP4 is located in the TZ

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To determine where NPHP4 is located in C. reinhardtii, we made an antibody to the C-terminus

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Accepted manuscript Journal of Cell Science

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of NPHP4. The antibody recognized a band of the expected size in western blots of wild-type

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but not nphp4 whole cells; the band was restored in the NPHP4-R strain (Fig. 1E). These results

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confirmed that NPHP4 is missing in the mutant. We also rescued the mutant with constructs

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expressing a 3xHA tag at either the N- or C-terminus of NPHP4 to generate NPHP4-HAN and

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NPHP4-HAC cells, respectively; the anti-HA antibody recognized the tagged protein with high

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specificity in the rescued cells (Fig. 1F). In immunofluorescence microscopy, the anti-NPHP4

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antibody strongly labeled the bases of the flagella of wild-type cells but not nphp4 cells (Fig. 2A),

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indicating that this localization is specific to NPHP4. Similarly, the anti-HA antibody localized

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specifically to the bases of flagella in both NPHP4-HAN and -HAC cells but not in wild-type

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cells, indicating that the localization is specific to HA-tagged NPHP4 (Fig. 2B).

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NPHP4 has been localized by immunofluorescence microscopy to primary cilia of

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MDCK and mouse kidney cells (Mollet et al., 2005; Patil et al., 2012). Therefore, we carefully

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determined the distribution of NPHP4 in C. reinhardtii flagella vs. cell bodies. When cells are

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deflagellated, the flagella detach at the junction of the TZ and axoneme proper (Sanders and

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Salisbury, 1989). NPHP4-HAC cells were deflagellated with dibucaine and the resulting cell

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bodies and detached flagella were stained with anti-HA; NPHP4-HAC stayed with the cell

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bodies (Fig. 2C). To confirm this result, whole cells, cell bodies, and isolated flagella were

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compared by western blotting; all of the NPHP4-HAC remained with the cell body and none

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could be detected in the isolated flagella (Fig. 2D). The results indicate that C. reinhardtii

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NPHP4 is located in either the TZ or basal body, but not in the flagella.

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To further narrow down the location of NPHP4, both nucleo-flagellar apparatuses and

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whole cells of the NPHP4-HAC strain were labeled with antibodies against the HA-peptide and

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CEP290, which is located in the TZ (Craige et al., 2010). By both wide-field

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immunofluorescence microscopy (Fig. 3A, B) and total internal reflection fluorescence/epi-

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fluorescence structured light microscopy (TESM) (Fig. 3C), the NPHP4-HAC label was distal to

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the CEP290 label, indicating that NPHP4 is located in the TZ. Analysis of TESM images

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indicated that the mean peak-to-peak distance between CEP290 and the C-terminus of NPHP4-

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HAC is about 137 nm (n=130).

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NPHP4 is located at the periphery of the distal TZ

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To determine the location of NPHP4 within the TZ, we carried out pre-embedding immuno-EM

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Accepted manuscript Journal of Cell Science

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of nucleo-flagellar apparatuses from wild-type and NPHP4-HAN and -HAC cells. As expected

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from the immunofluorescence microscopy results (Fig. 2B), gold particles were associated with

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the TZ in both NPHP4-HAN and -HAC specimens (Fig. 4 A, B). Most gold particles in

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longitudinal sections and all particles in cross sections of the TZ were located at the TZ periphery,

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in close association with remnants of the TZ membrane, which is resistant to treatment with

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nonionic detergent (Kamiya and Witman, 1984). We observed no gold particles associated with

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the wild-type TZs, indicating that the immuno-gold labeling was specific to HA-tagged NPHP4.

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The locations, relative to the proximal end of the TZ, of a total of 50 gold particles from

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longitudinal sections of both NPHP4-HAN and -HAC specimens were measured and compared

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to that determined for CEP290 by Craige et al. (2010) (Fig. 4C). Consistent with our

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immunofluorescence microscopy observations (Fig. 3), the distribution of both NPHP4-HAN

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and -HAC gold particles had sharp peaks in the distal TZ. The average distance of gold particles

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from the proximal end of the TZ was 173 ± 37 nm for NPHP4-HAC and 155 ± 31 nm for

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NPHP4-HAN, compared to 61 ± 38 nm for CEP290; the average distance between NPHP4-HAC

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and CEP290 gold particles was 111 nm, in good agreement with the separation (~137 nm)

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measured by TESM.

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NPHP4 and CEP290/NPHP6 localize to the TZ independently of each other

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To explore if C. reinhardtii NPHP4 can be assembled at the TZ independently from CEP290 and

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vice versa, we carried out immunofluorescence microscopy of cep290 and nphp4 mutant cells

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with antibodies against NPHP4 and CEP290, respectively. Localization of NPHP4 appeared to

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be normal in the cep290 cells, and localization of CEP290 was normal in the nphp4 cells

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(supplementary material Fig. S2A, B). Therefore, these two proteins are transported to and

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assemble into the TZ independently of each other.

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NPHP4 does not turnover rapidly

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We previously reported that CEP290 at the TZ is highly dynamic (Craige et al., 2010). To

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determine if NPHP4 is similarly dynamic, we mated wild-type gametes to NPHP4-HAN or

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NPHP4-HAC gametes to form quadriflagellated zygotes in which two TZs contained untagged

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NPHP4 and two contained HA-tagged NPHP4 in a common cytoplasm. Nucleo-cytoskeletons

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were prepared at 20, 40, and 60 min after initiation of the mating reaction and labeled with anti-

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HA (Fig. 5). Even after 60 min, only two TZs were labeled by the antibody, indicating that little

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if any HA-tagged NPHP4 was incorporated into the wild-type TZs over this time period.

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Moreover, the label was as bright at 60 min as at 20 min, indicating that little if any HA-tagged

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NPHP4 was replaced with wild-type NPHP4. Therefore, in contrast to CEP290, NPHP4 is static

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at the TZ.

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Ultrastructure of the TZ in the nphp4 mutant

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To determine if loss of NPHP4 caused structural abnormalities in the TZ, we compared the TZs

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of wild-type and nphp4 mutant cells by EM. In cross sections, the Y-links between the TZ

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doublet microtubules and membrane, which are disrupted in the cep290 mutant (Craige et al.,

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2010), were present and appeared normal (Fig. 6A). In longitudinal sections, the wedge-shaped

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connectors, which extend between the TZ doublets and membrane in wild-type cells, appeared to

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be missing or collapsed in the nphp4 mutant (Fig. 6B). However, the wedge-shaped structures

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are similarly altered in TZs of cells lacking IFT protein ift46 (Hou et al., 2007). To clarify if

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disruption of this structure is correlated with lack of NPHP4, we performed immunofluorescence

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microscopy of ift46 cells and found that NPHP4 was normally located at the base of their flagella

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(supplemental material Fig. S2C). Thus, the morphological variation in this structure is not

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specifically related to loss of NPHP4. Moreover, because the Y-links are present in the nphp4

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mutant, we can conclude that they and the wedge-shaped structures are not the same.

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Finally, in ~30% of longitudinal sections of nphp4 flagella, we observed, just distal to the

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TZ, the apparently ectopic location of dense material resembling the central cylinders of the C.

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reinhardtii TZ (Fig. 6C).

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Protein composition of the flagellar membrane and matrix is drastically altered by loss of

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NPHP4

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To compare the protein compositions of wild-type vs. nphp4 flagella, flagella were isolated and

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treated with NP-40 to separate proteins into a detergent-soluble membrane-plus-matrix fraction

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and an axonemal fraction. In SDS-polyacrylamide gels of the axoneme fractions, very few

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differences were observed (Fig. 7A, B). However, numerous differences were observed in the

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membrane-plus-matrix fractions. Some proteins were decreased in the nphp4 mutant

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(arrowheads, Fig. 7A) whereas others were increased (arrows, Fig. 7A). Normal flagellar protein

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composition was restored in NPHP4-R (supplemental material Fig. S3), confirming that the

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differences were due to loss of NPHP4. Importantly, most differences between the wild-type and

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nphp4 membrane-plus-matrix fractions were observed in the mass range above 50 kDa; few

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differences were observed below 50 kDa (Fig. 7B).

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To identify the proteins that were decreased or increased in the nphp4 flagella, sets of

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slices containing the discordant bands were excised from gels of the membrane-plus-matrix

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fraction of wild-type, nphp4, and NPHP4-R flagella (supplemental material Fig. S3). The slices

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were then subjected to analysis by mass spectrometry. Multiple proteins were identified in all

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slices (supplemental material Table S1). To conclude that the amount of a protein was altered in

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the mutant flagella, we used the following criteria: 1) the number of peptides recovered had to

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be increased or decreased by more than 10 compared to wild type, or the number of peptides

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recovered had to be greater than 5 when the protein was not found in the other slice (wild type or

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nphp4) being compared; and 2) the change was specifically rescued in the NPHP4-R cells.

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Based on these criteria, the proteins increased or decreased in the nphp4 mutant flagella are listed

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in Table I and supplemental material Table S1, where blue or red font indicates increased or

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decreased proteins, respectively. Except for ATPase V1 complex-subunit A, all increased

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proteins are housekeeping proteins that presumably are abundant in the cell bodies. Valyl- and

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phenylalanyl-tRNA synthetase, ADP ribosylglycohydrolase, and RNA-binding protein were not

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found in the C. reinhardtii flagellar proteome (Pazour et al., 2005). All increased proteins are

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predicted to be larger than 50 kDa (Table I). We conclude that large housekeeping proteins are

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specifically increased in nphp4 mutant flagella.

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On the other hand, all decreased proteins other than FAP208 are predicted to be

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membrane associated: the calcium ATPase is predicted to have a transmembrane domain, and

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flagellar adenylate kinase, FAP33, FAP295, and FAP12 have N-myristoylation sites.

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Additionally, when we examined other proteins also predicted to be membrane associated but not

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meeting the above criteria (these proteins were identified by less than 5 peptides in wild type,

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were not detected at all in the nphp4 mutant, and were restored in the rescued strain), all were

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decreased in the mutant flagella. These included the large flagella membrane glycoprotein

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FMG1-B, a predicted ion channel, another calcium transport ATPase, an ABC transporter, and

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FAP29, all of which have transmembrane domains; and ankyrin-repeat protein and apyrase,

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which contain N-myristoylation sites (yellow font in supplemental material Table S1). Nearly all

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decreased proteins were previously identified in the C. reinhardtii flagellar proteome (Pazour et

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al., 2005). Thus, proteins normally associated with the flagellar membrane are either not

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transported into or are not retained in the flagella in the absence of NPHP4.

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To determine if all flagella-associated membrane proteins are reduced in amount in nphp4

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flagella, we probed western blots of isolated flagella from wild type and the nphp4 mutant with

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antibodies against two well-characterized flagellar membrane proteins not identified in our

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dataset. The flagellar mastigoneme protein (Nakamura et al., 1996) and PKD2 (homologue of

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human polycystin-2; Huang et al., 2007) were not decreased in the nphp4 flagella (Fig. 7C). We

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also probed for FMG-1B, which appeared to be decreased in nphp4 flagella in our mass

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spectrometry analysis; the western blot confirmed that it was reduced in the mutant flagella. We

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conclude that only a subset of proteins normally associated with the flagellar membrane is

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decreased in the absence of NPHP4.

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IFT in the nphp4 mutant

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Several IFT proteins were identified by mass spectrometry and the levels of most of them did not

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seem to be altered in the mutant flagella (supplemental material Table S1). To confirm this, we

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examined the amounts of IFT components in isolated flagella of wild type and nphp4 by western

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blotting. The levels of IFT motors, of IFT proteins of both complex A and B, and of BBS4, a

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subunit of the BBSome, were unaltered or slightly elevated in the nphp4 mutant flagella

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(supplemental material Fig. S4A). To more directly assess if IFT is affected by lack of NPHP4,

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IFT particle movement in steady-state flagella was analyzed (supplemental material Fig. S4B).

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IFT frequency was not affected whereas IFT velocity was slightly decreased by the absence of

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NPHP4 (supplemental material Fig. S4C).

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DISCUSSION

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NPHP4 and CEP290 define distinct regions of the TZ

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In agreement with previous studies in C. elegans and mammals (Winkelbauer et al., 2005;

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Jauregui et al., 2008; Shiba et al., 2010; Sang et al., 2011; Williams et al., 2011), we found that

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C. reinhardtii NPHP4 is located in the TZ. However, our studies provide much more precise

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information on the distribution and localization of NPHP4. Using both immunofluorescence

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microscopy and immuno-EM, we determined that NPHP4 is located in the distal part of the TZ,

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distal to CEP290. The NPHP4 and CEP290 peaks observed by TESM were separated by a

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minimum of ~140 nm, and immuno-EM revealed that there is little overlap between the two

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proteins. Thus, NPHP4 and CEP290 define distinct structural domains of the TZ (Fig. 7D).

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Immuno-EM also indicated that the N- and C-termini of NPHP4 are located at the periphery of

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the TZ, in close association with the TZ membrane. Thus, NPHP4 is well positioned to control

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entry and/or exit of membrane proteins from the flagellum. This apparent localization of NPHP4

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to the extreme periphery of the TZ contrasts with that of CEP290, which is distributed

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throughout the space between the doublet microtubules and the membrane (Craige et al., 2010);

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however, we cannot rule out that the middle part of NPHP4 extends into the space between the

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membrane and microtubules. Finally, our biochemical studies of isolated flagella indicated that

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there is no detectable NPHP4 in the flagellum proper.

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Consistent with NPHP4 and CEP290 being located in different regions of the TZ, we

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found that NPHP4 and CEP290 each assembled normally into the TZ in the absence of the other.

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Both the localization and assembly results indicate that C. reinhardtii NPHP4 and CEP290 are

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integrated into different complexes in the TZ. These findings agree with those of Sang et al.

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(2011), who concluded that mammalian NPHP4 and CEP290 were components of distinct

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modules based on immunoprecipitations of LAP-tagged proteins.

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We also observed that NPHP4 at the TZ undergoes little or no turnover during a 1 h

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period; this is in sharp contrast to CEP290, which is at least 50% replaced in the same time span

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(Craige et al., 2010). Therefore, NPHP4 is static at the TZ, as might be expected for a structural

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protein. On the other hand, CEP290 appears to be much more dynamic. Although highly

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speculative, the results raise the possibility that CEP290 has a role in shuttling IFT or other

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ciliary proteins into the TZ.

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Accepted manuscript Journal of Cell Science

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NPHP4 and CEP290 loss have different consequences for TZ structure

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A highly conserved and defining feature of the TZ is the occurrence of Y-links between the

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doublet microtubules and membrane as observed in EM cross-sections of various organisms

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(Gilula and Satir, 1972; Perkins et al., 1986; Ringo, 1967). Craige et al. (2010) observed that the

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Y-links were missing or disrupted in C. reinhardtii CEP290 mutants. We found that loss of

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NPHP4, in contrast, did not affect the Y-links. Therefore, NPHP4 is not required for Y-link

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formation. The same is true in C. elegans, where loss of NPHP4 alone did not cause disruption

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of the Y-links (Jauregui et al., 2008). However, when mutations in NPHP4 and members of the

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MKS/B9 module were combined in C. elegans, the Y-links were lost (Williams et al., 2011).

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Although no specific structural abnormalities were observed in nphp4 TZs, the flagella

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often contained, distal to the TZ, what appeared to be a partial duplication of the central cylinder

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that normally occupies the lumen of the TZ of C. reinhardtii. Interestingly, a similar duplication

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of the central cylinder has been observed in uniflagellar mutants of this organism (Huang et al.,

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1982; Piasecki et al., 2008). The duplication may occur because the cell detects abnormalities

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associated with the absence of NPHP4 and responds by moving additional central cylinder

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precursors into the flagellum.

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A size-dependent barrier to ciliary entry of large soluble housekeeping proteins is breached

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in nphp4 cells

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Because of the ease with which C. reinhardtii flagella can be purified, we were able to assess the

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biochemical consequences of loss of NPHP4 for the flagella. Strikingly, nearly all of the

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differences observed were in the membrane-plus-matrix fraction, not the axonemal fraction. A

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large number of proteins were increased in the nphp4 flagella, and, with the possible exception

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of subunit A of the V1-ATPase, all these proteins were soluble housekeeping proteins. Most of

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these proteins were not detected at all in the membrane-plus-matrix fraction from wild-type

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flagella, suggesting that their presence in the nphp4 flagellum resulted from the breakdown of a

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mechanism that normally keeps them out of the organelle.

343

Importantly, abnormal entry of soluble cytoplasmic proteins into the nphp4 flagellum

344

was observed only for proteins that migrated above 50 kDa in SDS-PAGE, indicating that there

345

is an NPHP4-dependent barrier at the TZ that excludes soluble proteins of ~50 kDa or larger.

346

Consistent with this, Kee et al. (2012) observed that when fluorescently labeled protein A (41

12

Accepted manuscript

347

kDa) and bovine serum albumin (67 kDa) were microinjected into hTERT RPE cells, the former

348

entered the ciliary compartment, whereas the latter did not. Similarly, Breslow et al. (2013),

349

working with permeabilized IMCD3 cells, found that the rate of entry of soluble proteins into

350

the cilium progressively slowed as the proteins increased in size from a Stokes radius of 3.1 nm

351

to 4.3 nm (corresponding to ~30 to ~70 kDa); no entry was observed for larger proteins. Taken

352

together, these results indicate that there is a size-dependent barrier to diffusion of soluble

353

proteins into the cilia of organisms ranging from protists to humans and that this barrier is

354

dependent upon NPHP4 in the TZ.

355

Using a chemically inducible diffusion trap approach, Lin et al. (2013) also obtained

356

evidence for a size-dependent diffusion barrier at the base of primary cilia. However, they

357

observed slow entry of proteins as large as ~600-650 kDa, and suggested that “the steady-state

358

distribution of proteins in cilia is unlikely to be regulated by the diffusion barrier.” Because the

359

differences we observed were in steady-state flagella that were at least 3 hours old, the C.

360

reinhardtii NPHP4-dependent diffusion barrier appears to be more impermeable to the diffusion-

361

based entry of large soluble proteins.

Journal of Cell Science

362 363

NPHP4 is essential to a TZ barrier that retains a subset of membrane proteins in the

364

flagella

365

While levels of large soluble housekeeping proteins increased in nphp4 flagella, levels of many

366

membrane-associated proteins decreased. However, the levels of some other flagellar membrane

367

proteins, such as PKD2 and the mastigoneme protein, were not affected in nphp4 flagella. C.

368

reinhardtii PKD2 is likely to be anchored to the axoneme because most PKD2 peptides were

369

found in axonemal fractions in the flagellar proteome (Pazour et al., 2005) and more than 90% of

370

GFP-tagged PKD2 was stationary in the flagella (Huang et al., 2007). Similarly, 75% of

371

peptides from the mastigoneme protein identified in the flagellar proteome were found in the

372

fraction resistant to detergent, implying that this protein also is anchored to the axoneme (Pazour

373

et al., 2005). These results agree well with reports that PKD2 and an odorant receptor are

374

normally located in C. elegans nphp-4 cilia (Jauregui et al., 2008; Williams et al., 2011). We

375

further observed that the level of FMG-1B was decreased; in the C. reinhardtii flagellar

376

proteome, peptides from FMG-1B were evenly distributed between the membrane and axonemal

377

fractions (Pazour et al., 2005), and at least some of this protein is moved in the plane of the

13

Accepted manuscript Journal of Cell Science

378

flagellar membrane by IFT (Guilford and Bloodgood, 2013). Taken together, these results

379

suggest that a subset of flagellar membrane proteins – specifically those presumed to be mobile

380

in the plane of the membrane – are reduced in nphp4 flagella, whereas those that are anchored to

381

the axoneme exhibit no reduction. Because PKD2 and the mastigoneme protein that bind to the

382

axoneme appear to be delivered normally to the nphp4 flagella, it is possible that most or all

383

flagellar membrane proteins are targeted normally to the flagella, but those that are free to move

384

in the plane of the membrane subsequently diffuse out of the organelle in the absence of an

385

NPHP4-dependent barrier at the TZ.

386

Previous studies have provided evidence for a membrane protein barrier at the base of the

387

cilium that is consistent with our findings. Studies with mammalian cells showed that disruption

388

of members of the MKS/B9 complex, which localizes to the TZ, resulted in a decrease in the

389

amount of several GFP-tagged membrane proteins in primary cilia (Garcia-Gonzalo et al., 2011;

390

Chih et al., 2012). NPHP4 is not part of the MKS/B9 complex, but studies in C. elegans indicate

391

that NPHP-4 and proteins of the MKS/B9 complex genetically interact in functionally redundant

392

pathways important for ciliary development (Williams et al., 2008, 2010, 2011; Huang et al.,

393

2011; Warburton-Pitt et al., 2012). Therefore, NPHP4 and the MKS/B9 proteins likely function

394

together to prevent ciliary membrane proteins from diffusing out of the cilium. NPHP4 is

395

located close to or at the TZ membrane and several of the MKS/B9 proteins are predicted to be

396

transmembrane proteins, so it will be of interest to determine precisely where the MKS/B9

397

proteins are located relative to NPHP4 and CEP290 in the TZ.

398 399

Loss of NPHP4 has mild effects on flagellar motility

400

Interestingly, the only behavioral defect that we observed in the nphp4 mutant was erratic

401

swimming. The lack of a stronger motility defect is consistent with our finding that there is little

402

if any change in the protein composition of the flagellar axoneme, which constitutes the motile

403

machinery. However, changes in intraflagellar Ca2+ are critical for controlling flagellar

404

waveform (Bessen et al., 1980; Kamiya and Witman, 1984), so the erratic swimming that we

405

observed is entirely consistent with the loss of membrane proteins such as the Ca2+-transport

406

ATPases, which are likely involved in maintaining normal intraflagellar Ca2+ levels.

407 408

NPHP4 and CEP290 define different structural and functional domains in the TZ

14

Although nphp4 cells are able to assemble full-length flagella, the kinetics of flagellar

410

regeneration following flagellar amputation is significantly impaired. Presently the reason for

411

this is unclear. IFT frequency is normal and velocity is only slightly reduced in steady-state

412

nphp4 flagella, suggesting that loss of NPHP4 does not directly affect the IFT machinery. These

413

results are generally consistent with those in C. elegans, where mutation of NPHP-4 has modest

414

(Jauregui et al., 2008) or no (Williams et al., 2011) effect on IFT. The slower regeneration

415

kinetics that we observed may reflect reduced IFT cargo loading, impaired flagellar signaling

416

necessary for normal regeneration, or altered intraflagellar ion concentrations (see above

417

section). In any case, the very slight effect of NPHP4 loss on IFT is in contrast to the effect of

418

CEP290 loss, which results in severe IFT abnormalities and greatly impaired flagellar formation

419

(Craige et al., 2010). Therefore, although NPHP4 and CEP290 are both essential to the TZ

420

barrier, CEP290 has an additional function in IFT. We conclude that NPHP4 and CEP290 define

421

different functional as well as structural domains in the TZ.

422

Journal of Cell Science

Accepted manuscript

409

15

423

MATERIALS AND METHODS

Journal of Cell Science

Accepted manuscript

424 425

Strains and growth medium

426

C. reinhardtii wild-type strains 137c (nit1, nit2, mt+) and CC124 (nit1, nit2, mt-) were obtained

427

from the Chlamydomonas Resource Center (University of Minnesota, St. Paul, MN). B1179

428

(nphp4-1, drc3-1, arg7, ARG7, mt+) was generated by insertional mutagenesis of H11 (arg7,

429

mt+) with the pARG7.8 plasmid (Debuchy et al., 1989). The nphp4 strain 79D8 (nphp4-1, drc3,

430

DRC3, mt+), rescued for DRC3, was produced by crossing B1179 sequentially to the wild-type

431

strains CC124 and 137c and then transforming selected progeny with DRC3. NPHP4-R (nphp4-

432

1, NPHP4, drc3, DRC3, mt+) was made by transformation of 79D8 with NPHP4. Two kinds of

433

HA-tagged NPHP4 strains were created: NPHP4-HAN (nphp4-1, NPHP4-HAN, drc3, DRC3,

434

mt+) was generated by transformation of 79D8 with NPHP4-HAN. NPHP4-HAC (nphp4-1,

435

NPHP4-HAC, drc3, DRC3, mt-) was made by transformation of B1179 with NPHP4-HAC

436

followed by two crosses to wild-type strains (CC124 and 137c, sequentially) and transformation

437

of the offspring with DRC3. Cells were grown in M medium I (Sager and Granick, 1953)

438

modified with 2.2 mM KH2PO4 and 1.7 mM K2HPO4 (Witman, 1986) or TAP medium (Gorman

439

and Levine, 1965).

440 441

Real time PCR

442

The deletion site of B1179 was determined by real-time PCR using a thermal cycler (Opticon,

443

MJ Research), the QuantiTect SYBR Green PCR kit (Qiagen), and primers listed in

444

supplemental material Table S2.

445 446

Gene cloning and transformation

447

To clone the full length NPHP4 gene, a 7,159-bp HindIII-SpeI fragment and a 6,357-bp SpeI

448

fragment were cut from BAC clone 18L19 containing NPHP4 and inserted sequentially into

449

pBluescript II SK- (Stratagene). To generate a construct encoding NPHP4-HAN having three

450

consecutive HA peptides (3xHA tag) between the first and second amino acids of NPHP4, a

451

SmaI-NaeI fragment of the p3xHA plasmid (Silflow et al., 2001) was inserted into the AfeI site

452

of NPHP4. To generate a construct encoding NPHP4-HAC having a 3xHA tag between the

453

penultimate and last amino acids, a StuI-SmaI fragment from the p3xHA plasmid was subcloned

16

454

into the PsiI site of NPHP4. To clone DRC3, a 2,225-bp BamHI-SacI fragment and a 5,144-bp

455

SacI fragment were cut from BAC clone 34B19 and inserted into pBluescript II SK-. All BAC

456

clones were purchased from Clemson University Genomics Institute.

Journal of Cell Science

Accepted manuscript

457

All transformations were performed by the glass beads method (Kindle, 1990). For

458

transformation with wild-type and HA-tagged NPHP4, cells were co-transformed with the ble

459

gene as a selectable marker (Stevens et al., 1996). Colonies on TAP medium plates containing

460

zeomycin were randomly selected and screened by immunofluorescence microscopy with anti-

461

NPHP4 or anti-HA antibodies. For transformation with DRC3, the aph7’’ gene was used as the

462

selectable marker (Berthold et al., 2002).

463 464

Analysis of motility

465

Cultures of 137c, 79D8, and NPHP4-R cells at 104-105 cells/ml were diluted 5-10 times with

466

modified M medium for motility analysis. To make a 100-μm deep chamber for observation of

467

the cells, 22 x 22 and 24 x 30 mm No. 1 coverslips were glued to opposite ends of a 25 x 75 mm

468

slide glass so that the two coverslips were separated by about 23 mm. An aliquot (~20 μl) of the

469

cell culture was then placed between the coverslips and covered with a 2X-CEL cover glass

470

(Hamilton Thorne Research). The chamber was inserted into an IVOS sperm analyzer (version

471

12; Hamilton Thorne Research) and cell movement recorded using dark-field optics and a 5x

472

objective.

473 474

Antibodies

475

The last exon of C. reinhardtii NPHP4 was amplified by PCR with primers NPHP4Ex28-N and -

476

C linked to BamHI and SalI sites, respectively. The amplified DNA fragment was cut with those

477

enzymes and put into pMAL-cRI (New England Biolabs) to express the last exon of NPHP4

478

fused to maltose-binding protein (MBP). The fusion proteins were purified with amylose resin

479

and used to produce rabbit polyclonal antibodies (Covance). The NPHP4 antibodies were

480

purified by affinity chromatography with the antigen attached to agarose resin. Bound antibodies

481

were eluted and applied to an agarose column containing MBP to remove anti-MBP antibodies;

482

the flow-through fraction was used in this study. Other antibodies used were anti-βF1-ATPase

483

(Atteia et al., 2006); anti-HA peptide (3F10, Roche); anti-acetylated tubulin (Sigma-Aldrich);

484

anti-α-tubulin (Silflow and Rosenbaum, 1981); anti-CEP290 (Craige et al., 2010); anti-FMG1-B

17

485

(Bloodgood et al., 1986); anti-mastigoneme (Nakamura et al., 1996); anti-PKD2 (Huang et al.,

486

2007); anti-KAP (Mueller et al., 2005); anti-DHC1b (Pazour et al., 1999); anti-IC2 (King et al.,

487

1985); anti-BBS4 (Lechtreck et al., 2009); anti-IFT46 (Hou et al., 2007); and anti-IFT57, anti-

488

IFT81, anti-IFT139, and anti-IFT172 (Cole et al., 1998).

Journal of Cell Science

Accepted manuscript

489 490

Immunofluorescence microscopy

491

Cells were applied for 1-3 min to a coverslip coated with polyethyleneimine, the excess cell

492

suspension removed by blotting, and the coverslip immersed in chilled methanol (-20°C) for 15

493

min. The coverslip was then completely dried before treating with blocking solution (3% fish

494

gelatin, 1% BSA in PBS pH 7.0) for 30 min at RT. Primary antibody diluted in the blocking

495

solution was applied to the sample for overnight at 4°C, followed by 4 washes with PBS.

496

Secondary antibodies diluted in the blocking buffer were then put on the coverslip for 1 h at RT.

497

For double staining, secondary antibodies conjugated to Alexa 488 and 568 or 594 (Life

498

Technologies Corp.) were used. For tri-color labeling, secondary antibodies conjugated to Alexa

499

350, 488, and 594 were used. Finally, the coverslip was rinsed 3 times for 10 min with PBS and

500

mounted on a glass slide with ProLong Gold Antifade Reagent (Life Technologies Corp.).

501

To prepare nucleo-flagellar apparatuses, NPHP4-HAC cells were treated with autolysin,

502

resuspended in ice-cold microtubule-stabilizing (MT) buffer (30 mM Hepes-KOH pH 7.4, 5 mM

503

MgSO4, 15 mM KCl, 2 mM EGTA), and mixed with an equal volume of 1% NP-40

504

(Calbiochem) in MT buffer. The solution was left on ice for 15 min and then mixed with an

505

equal volume of 6% paraformaldehyde in MT buffer. After incubation on ice for 60 min, the

506

nucleo-flagellar apparatuses were collected by centrifugation at 1,100 x g for 10 min. The

507

cytoskeletons were attached to a coverslip coated with poly-L-lysine and dried. The samples

508

were blocked, labeled, and mounted as described above for whole cells.

509

The microscope, camera, and software used to take fluorescence images were described

510

previously (Craige et al., 2010). Gamma adjustment and creation of merged images were done

511

with Photoshop CS2; all images mounted together were processed similarly.

512 513

TESM

514

TESM images were taken using our Biomedical Imaging Group's custom built TESM system

515

(Navaroli et al., 2012). Cells triple labeled with Alexa 488, 594, and 647 and mounted in

18

Accepted manuscript Journal of Cell Science

516

ProLong Gold Antifade Reagent were imaged using three color epi-fluorescence illumination.

517

The pixel size corresponded to 0.083 microns. Z-stacks consisted of 100 images with a z

518

separation of 100 nm. At each z-plane an exposure of 25 ms was taken in each emission

519

bandpass.

520

Tetraspec beads (200 nm) were used to measure chromatic aberration in the microscope

521

system. Point spread functions fitted to the beads in each color revealed a chromatic aberration

522

between the green and the red and far red channels of 1.6 pixels in the x direction. This

523

measured offset was used to correct the experimental data.

524 525

EM

526

Whole cells were pre-fixed in 1% glutaraldehyde in modified M medium for 15 min at RT,

527

collected by centrifugation, resuspended with 1% glutaraldehyde in 100 mM sodium cacodylate-

528

NaOH pH 7.2 (cacodylate buffer), and left for 2 h at RT. Specimens were then processed for

529

ultra-thin sectioning as described previously (Craige et al., 2010).

530

For pre-embedding immunogold EM, nucleo-flagellar apparatuses of 137c, NPHP4-HAN,

531

and NPHP4-HAC cells were prepared as described in “Immunofluorescence microscopy.” After

532

fixation with 3% paraformaldehyde, the samples were washed three times with PBS + 0.02%

533

Tween-20 (PBST) and blocked with 5% skin milk, 4% BSA in PBST for 1 h at RT. The

534

cytoskeletons were then resuspended with the anti-HA antibody diluted 1:400 in PBST and

535

incubated overnight at 4°C. After three washes with PBST, the samples were next incubated

536

with 1% BSA in PBST containing anti-rat IgG conjugated with 10-nm gold particles (Aurion).

537

The cytoskeletons were then washed three times in MT buffer and fixed with 1% OsO4 in MT

538

buffer for 30 min at RT. The pellets were then washed once with MT buffer, rinsed three times

539

with distilled water, and stained with 1% uranyl acetate overnight at 4°C. Dehydration and

540

embedding was performed as for whole cells, except that post-sectioning staining was omitted.

541 542

Generation of gametes and dikaryons

543

To produce gametes, 137c, CC124, NPHP4-HAN, and NPHP4-HAC cells grown to about 105

544

cells/ml in 125 ml of M medium I were collected and resuspended twice with 15 ml of nitrogen-

545

free M medium. The second suspensions were transferred to 250-ml flasks and agitated under

546

constant light overnight. Quadriflagellated zygotes were generated by mixing 137c with

19

547

NPHP4-HAC and CC124 with NPHP4-HAN gametes. At various times after initiation of the

548

mating reaction, aliquots of the mixture were removed and nucleo-flagellar apparatuses prepared

549

as described under “Immunofluorescence microscopy.”

Journal of Cell Science

Accepted manuscript

550 551

Flagella isolation, SDS-PAGE, and western blotting

552

Flagella were isolated by the dibucaine method (Witman, 1986) with the following

553

modifications: DTT was omitted from the HMDS solution. The isolated flagella were

554

resuspended with HMDEK buffer containing 30 mM Hepes pH 7.4, 5 mM MgSO4, 1 mM DTT,

555

1 mM EGTA, and 25 mM KCl. To obtain axonemal and membrane-plus-matrix fractions, NP-40

556

was added to the flagellar suspension to a final concentration of 1% and the suspension left on

557

ice for 10 min. The suspension was then centrifuged (26,890 x g for 10 min at 4°C) and the

558

supernatant collected as the membrane-plus-matrix fraction. The pellet containing axonemes

559

was resuspended in HMDEK buffer. For SDS-PAGE, each fraction was mixed with one-fourth

560

volume of 5xSDS-sample solution (0.3 M Tris-HCl pH 6.8, 5 mM EDTA, 10% SDS, 25%

561

sucrose, and 0.01% bromophenol blue), and DTT added to a final concentration of 50 mM. The

562

mixture was then incubated at 75°C for 20 min. Protein bands were visualized with silver stain

563

(Silver Stain Plus; Bio-Rad). To prepare whole flagella for western blotting, a suspension of

564

isolated flagella was mixed with SDS-sample solution and DTT as described above. For whole

565

cells, cells were collected by centrifugation, lysed with the 5xSDS-sample solution, and

566

incubated at 75°C for 20 min in the presence of 50 mM DTT. Proteins in SDS-polyacrylamide

567

gels were transferred onto Immobilon-P (Millipore). For western blots of whole cells, antibodies

568

were diluted with HIKARI (Nacalai Tescue, Inc.). The chemiluminescence or fluorophore

569

signals from the secondary antibodies were captured with a FluorChem Q equipped with a CCD

570

camera (ProteinSimple).

571 572

Mass spectrometry

573

Protein bands of interest were excised from silver-stained gels and subjected to trypsin digestion

574

as described by Spiess et al. (2011). Mass spectrometry analysis was performed as described by

575

Wagner et al. (2014) with the following modifications: length of LC column was 15 cm and the

576

flow rate of sample injection was 250 nl/min. The resulting spectra were searched against the C.

577

reinhardtii Augustus 5 protein database (Erik Hom, personal communication; based on the Joint

20

578

Genome Institute’s Assembly 4 of the C. reinhardtii genome: http://genome.jgi-

579

psf.org/Chlre4/Chlre4.home.html) using SEQUEST (Bioworks software, v3.3.1; Thermo

580

Electron).

Journal of Cell Science

Accepted manuscript

581 582

Flagella regeneration

583

To determine the kinetics of flagellar regeneration, the pH of mid-log phase cultures of 137c and

584

79D8 cells was lowered to 4.3 with 0.5 M acetic acid. As soon as deflagellation was confirmed

585

by microscopy, the pH was raised to 6.9 with 0.5 M KOH. To fix cells at each time point, a

586

small aliquot of the cell suspension was mixed with an equal volume of M medium containing

587

2% glutaraldehyde. Flagella lengths were measured with the Segmented Line tool in ImageJ

588

(National Institutes of Health) and mean values at each point were obtained from 50 flagella on

589

25 cells.

590 591

Observation of IFT

592

137c and nphp4 cells at about 104 cells/ml were used to observe IFT as previously described

593

(Craige et al., 2010). Kymographs were made by ImageJ with the MultipleKymograph plugin.

594

21

595

ACKNOWLEDGEMENTS

596

We are grateful to all those who generously provided antibodies, to the University of

597

Massachusetts Medical School (UMMS) Core EM Facility for expert help with EM, and to the

598

Vermont Genetics Network (VGN) Proteomics Facility for mass spectrometry. We thank Eric

599

Coleman Johnson for identifying the nphp4 mutant. We also thank Erik Hom (Harvard

600

University) for providing C. reinhardtii Augustus 10 gene models, and Branch Craige (UMMS)

601

for critical discussion.

Accepted manuscript

603

609

Funding

Journal of Cell Science

602

610

This research was supported by NIH grants GM060992 (to GJ) and GM030626 (to GW), by the

611

Robert W. Booth Endowment at UMMS (to GW), and by NIGMS Institutional Development

612

Award (IDeA) P20 GM103449 (to the VGN).

Author contributions

604

J.A. designed and performed most experiments. S.T. did the initial characterization of the nphp4

605

mutant. C.S., K.D.B., and K.E.F. acquired data with TESM. K.F.L. assisted with some

606

experiments. J.A. and G.B.W. prepared the manuscript. G.J.P and G.B.W. supervised and

607

funded this study and helped interpret the data.

608

613 614

22

615

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Journal of Cell Science

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FIGURE LEGENDS

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Figure 1. Characterization of C. reinhardtii nphp4 mutant. (A) Map of C. reinhardtii

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genome near the NPHP4 locus. Numbers above each locus correspond to gene IDs in

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Phytozome version 9.1 (http://www.phytozome.net). Arrows indicate the positions of PCR

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products used to delimit the deleted region; plus and minus marks indicate whether the PCR

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products were amplified. Genome fragments from bacterial artificial chromosome (BAC) clones

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used for knock-in of full-length NPHP4 and DRC3 are indicated by white rectangles. (B)

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Representative images showing swimming paths of wild-type and nphp4 cells. White dots show

Accepted manuscript

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cells where CASA began to monitor their tracks; green lines are the swimming paths analyzed.

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Blue tracks were not analyzed because the cells swam outside the microscope field before

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recording was completed. Cells marked with red dots were immobilized by attachment to the

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coverslip. The swimming paths of nphp4 cells are more erratic than those of wild-type cells. (C)

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Means ± s.d. of linearity and CASA histogram summarizing distribution of linearity in

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swimming paths of populations of wild-type, nphp4, and NPHP4-R cells (see supplementary

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material Fig. S1). For each strain, the population was calculated from the sum of values from a

Journal of Cell Science

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total of 10 fields in each of 5 independent experiments. Statistical significance was determined

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by the Tukey-Kramer method: **,