Transient Internalization and Microtubule-Dependent Trafficking of a Ciliary Signaling Receptor from the Plasma Membrane to the Cilium

Summary

Cilia are ancient organelles used by unicellular and multicellular organisms not only for motility, but also to receive and respond to multiple environmental cues, including light, odorants, morphogens, growth factors, and contact with cilia of other cells. Much is known about the cellular mechanisms that deliver membrane proteins to cilia during ciliogenesis. Execution of a ciliary signaling pathway, however, can critically depend on rapid alterations in the receptor composition of the cilium itself, and our understanding of the mechanisms that underlie these rapid, regulated alterations remains limited [1–6]. In the bi-ciliated, unicellular alga Chlamydomonas reinhardtii, interactions between cilia of mating type plus and mating type minus gametes mediated by adhesion receptors SAG1 and SAD1 activate a ciliary signalling pathway [7]. In response, a large, inactive pool of SAG1 on the plasma membrane of plus gametes rapidly becomes enriched in the peri-ciliary membrane and enters the cilia to become active and maintain and enhance ciliary adhesion and signalling [8–14]. Ciliary entry per se of SAG1 is independent of anterograde intraflagellar transport (IFT) [13], but the rapid apical enrichment requires cytoplasmic microtubules and the retrograde IFT motor, dynein 1b [14]. Whether the receptors move laterally within the plasma membrane or transit internally during redistribution is unknown. Here, in coupled immunolocalization/biochemical studies on SAG1, we show that within minutes after gamete activation is initiated, cell surface SAG1 is internalized, associates with an apico-basally polarized array of cytoplasmic microtubules, and returns to the cell surface at a peri-ciliary staging area for entry into cilia.

eTOC blurb

Ranjan et al. identify a pathway for regulated transit of ciliary receptors from the plasma membrane to the ciliary membrane in the bi-ciliated alga Chlamydomonas. Within minutes after signaling is triggered, receptors are internalized from the plasma membrane, align along cytoplasmic microtubules, and re-appear on the membrane at the ciliary base.

Results

during regulated trafficking to the ciliary membrane

When plus and minus gametes are mixed together and adhesion receptors SAG1 and SAD1 interact [15], the consequent activation of a protein kinase-dependent ciliary signaling pathway leads to increased levels of intracellular cAMP [7, 16–19]. Previous work using sag1 mutant plus gametes [15] rescued for adhesion with an epitope-tagged form of SAG1, SAG1-HA, demonstrated signaling-induced rapid redistribution of SAG1-HA from its presence over the entire plasma membrane to a highly polarized pattern with SAG1-HA enriched in the peri-ciliary region and in the cilia [13, 14], but those studies did not address the trafficking route.

Although cilia of naive gametes (i. e., gametes that had not yet experienced increased intracellular cAMP) have sufficient SAG1 to bring about initial ciliary adhesion and signaling, these low, sentinel levels in the organelles are difficult to detect by immunofluorescence (Figure 1A, left panels), but are detectable by immunoblotting of isolated cilia [13 and see below]. Figure 1A (right panels) documents the apically localized and ciliary enriched SAG1-HA in gametes incubated for 10 minutes in medium containing a cell-permeable form of cAMP, dibutyryl cAMP (db-cAMP) and a phosphodiesterase inhibitor. Closer examination of the confocal images of naive gametes (Figure 1B, left panels) showed that SAG1-HA was present as puncta distributed relatively evenly over the cell surface. Staining was not present in any discernible pattern on the plasma membrane, nor was it concentrated near the bases of the cilia. In the db-cAMP-treated gametes, however, altered patterns of SAG-HA distribution were apparent (Figure 1B, right panels). In some cells, the protein had become apically enriched (arrowheads) as reported previously. Notably, though, in some gametes SAG1-HA had not yet become polarized, but it appeared to have become internalized (asterisks).

Figure 1. Regulated polarization and internalization of SAG1 during signaling.

(A and B) Immunofluorescence (upper panels) and bright field (lower panels) confocal images of SAG1-HA in naive gametes and in gametes activated by incubation for 10 minutes (A) or 5 minutes (B) in medium containing db-cAMP and papaverine. Images show maximum-intensity projections of three z-slices that include the cilia. (Arrowheads indicate gametes with apical enrichment of SAG1-HA and asterisks indicate gametes in which ring staining was lost, but apical enrichment had not yet occurred. (C) Naive SAG1-HA gametes and gametes incubated for 5 minutes in db-cAMP were subjected to the protease sensitivity assay followed by immunoblotting for HA and tubulin. 2 × 10⁶ cells were loaded in each lane.

In a separate, biochemical approach to determine whether SAG1 had been internalized, we used a protease sensitivity assay [11, 20]. Surface localized SAG1-HA is degraded by trypsin treatment, whereas internalized SAG1-HA would be resistant. Consistent with previous studies [13], immunoblotting of the trypsin-treated, naive gametes showed that the majority of SAG1-HA was sensitive to protease treatment of live cells (Figure 1C), indicating that most was on the cell surface. On the other hand, analysis of gametes that had been incubated for 5 minutes in medium containing db-cAMP yielded the striking result that substantial amounts of the SAG1-HA had become resistant to the protease treatment (Figure 1C). Thus, cell surface SAG1-HA indeed was internalized during gamete activation.

SAG1-HA internalization is transient

SAG1 ultimately functions in adhesion and signaling on the surface of the ciliary membrane, and thus the internalized protein must return to the cell surface at some point during trafficking.

To learn more about the location of SAG1-HA during its trafficking to cilia, we assayed its accessibility to trypsin at increasing times of incubation of cells in db-cAMP. Remarkably, subsequent to its internalization at ~5 minutes, some SAG1-HA again began to be accessible to the protease at 15 minutes, and by 20 minutes, all detectable SAG1-HA was sensitive again (Figure 2A and S1). In validation of the assay, we determined that cytoplasmic proteins clathrin and tubulin were resistant to protease treatment of the live gametes (Figure 2A, lower panels); whereas, as expected, SAG1-HA and tubulin in detergent lysates were sensitive to the protease (Figure S1). Immunofluorescence microscopy of cells that had been incubated in db-cAMP for 20 minutes showed that on many cells it had become highly enriched in the peri-ciliary region and on the cilia (Figure 2B), results consistent with cell fractionation and immunoblotting of gametes activated for 30 minutes (Figure 2C). Thus, en route to the cilia, SAG1 was transiently internalized and then returned to the surface for ciliary entry.

Figure 2. SAG1-HA internalization is transient.

(A) Immunoblots of gametes (2 × 10⁶ cells per lane) incubated for the indicated times in db-cAMP followed by incubation with or without protease. Ponceau staining documents equivalent loading. Clathrin and tubulin immunoblots document that cytoplasmic proteins were inaccessible to trypsin. (B) Maximum intensity projections of immunofluorescence images of gametes incubated for 20 minutes in db-cAMP and stained for HA, a time when all SAG1-HA had again become sensitive to trypsin. (C) Immunoblot analysis of cell bodies and isolated cilia from naive gametes and gametes incubated in db-cAMP for 30 minutes (2.5 ug of protein were loaded in each lane; see also Figure S1).

Internalized SAG1-HA aligns along microtubules

We used immunofluorescence microscopy to determine the route followed by internalized SAG1-HA during transit from the surface of the cell plasma membrane to the peri-ciliary region. In lateral views of many cells after 10 minutes in db-cAMP, a time when the majority of total cellular SAG1-HA was internal, the protein was present in linear arrays that coalesced into fewer, more brightly stained structures near the apical end of the cells (Figure 3A, upper and lower panels; Video S1). Examination of images of db-cAMP-treated gametes viewed from the apical end (down the long axis) provided more information about the distribution of SAG1-HA. In addition to being enriched on the cilia, SAG1-HA was also concentrated near the bases of the cilia — the location of the basal bodies — into a striking, cruciate pattern (Figure 3B; Video S2). The insets show single optical sections that do not include the cilia, in which the cruciate pattern was most apparent, at the location of the basal bodies.

Figure 3. SAG1-HA localization soon after internalization.

(A) Bright field (left) and immunofluorescence (right) longitudinal images of maximum intensity projections of gametes incubated in db-cAMP for 10 minutes (also see Video S1) and stained for HA. (B) Maximum intensity projection of cross-sectional z-stacks. The insets show single z-stacks just below the level of the cilia in the adjacent cells (See also Videos S1 and S2).

The mid-cell, linear arrays and the apical, cruciate arrangements of SAG1-HA are hallmarks of the two sets of microtubules that compose the non-ciliary microtubule cytoskeleton in Chlamydomonas [21–27]. Both sets originate near the basal bodies and extend their plus ends basally. One set, termed the cytoplasmic or cortical microtubules, is present as individual, highly dynamic, singlet microtubules. They typically number between 10–20 and many extend fully to the basal end of the cell [21, 22, 24, 26–28]. Because they are approximately evenly distributed, these singlet, cytoplasmic microtubules form a cage-like structure enclosing the cytoplasm (Figure 4A-i; Video S3). The other set of microtubules, termed the root or rootlet microtubules, is composed of 4 bundles of much more stable microtubules – 2 bundles of two singlet microtubules and 2 bundles of 4 singlet microtubules. At their sites of origin between the basal bodies, the bundles are present in a cruciate pattern (Figure 4A-ii and B; Video S4), and each bundle extends to nearly the mid-point of the cell [21, 24–27]. (Whether some of the singlet microtubules in the bundles separate and continue to project to the basal end of the cell is unknown.)

Figure 4. Internalized SAG1-HA co-localizes with cytoplasmic microtubules.

(A) Confocal images showing tubulin staining of gametes incubated in db-cAMP for 10 minutes. (A-i) Maximum intensity projection of z-stacks captured from a lateral view (from Video S3). (A-ii) Maximum intensity projection of z-stacks taken down the long axis of the cell (from Video S4). (B) Schematic illustration of a cross-sectional view of the bundles of rootlet microtubules. (C) Maximum intensity projections of HA and tubulin staining in gametes activated for 10 minutes in db-cAMP (from Video S5). The right-most panels in each row are higher magnification views. Arrowheads indicate cell regions with long stretches of SAG1-HA aligned along microtubules. (See also Videos S3–S5).

In light of this microtubule distribution and our previous studies showing that the rapid apical localization of SAG1 during gamete activation required cytoplasmic microtubules and the retrograde IFT motor, cytoplasmic dynein 1b [13, 14], we assessed the relationship between SAG1 and microtubules. Examination of activated gametes co-stained with anti-HA and anti-tubulin antibodies indeed showed that the SAG1 was present on microtubules (Figure 4C and Video S5). In the mid-regions of gametes incubated for 10 minutes in db-cAMP buffer, the linearly organized strands of SAG1-HA were co-localized with microtubules (arrowheads, right-most panels). With the methods used, it was not possible to distinguish singlet microtubules from microtubule bundles, although it was likely that the prominent, tubulin-containing structures that co-stained for SAG1 at the apical ends of the cells were the bundled rootlet microtubules.

Discussion

In Chlamydomonas, gamete recognition and initiation of signaling occur exclusively in the cilia. Moreover, adhesion itself triggers inactivation of paired ciliary adhesion receptors SAG1 and SAD1 [8, 29], which are ultimately released in the form of ciliary exosomes [14]. Thus, to maintain adhesion and ciliary signalling until interacting plus and minus gametes fuse, SAG1 is delivered to the organelles from a pool on the surface of the plasma membrane. We had previously shown that although SAG1 entry per se into the cilia during signalling was independent of the anterograde IFT motor FLA10, ciliary adhesion-induced redistribution of SAG1 from the cell plasma membrane to the ciliary bases depended on cytoplasmic microtubules [13] and the retrograde IFT microtubule motor, cytoplasmic dynein 1b [14]. In the current work, we examined the cellular disposition of SAG1 during redistribution and discovered that it is internalized almost immediately after signaling is triggered. Furthermore, the internalized SAG1 co-localizes with the cytoplasmic arrays of microtubules; soon thereafter, it re-appears on the cell surface to become enriched near the bases of the cilia and on membrane of the organelles.

Based on our current findings and on previous studies, we favor the following model for redistribution of SAG1 triggered by cilium-generated signaling. The increase in intracellular cAMP induced by ciliary adhesion triggers endocytosis of SAG1 from the plasma membrane. The resulting SAG1-containing vesicles are carried by the minus-end directed retrograde IFT motor cytoplasmic dynein 1b along the cytoplasmic microtubules from their plus ends at the basal end of the cell towards their minus ends near their sites of origin at the basal bodies. Much like “All roads lead to Rome,” the cortical microtubules and the bundled rootlet microtubules converge at the basal bodies, and consequently the SAG1-containing vesicles are delivered to this site where they ultimately fuse with the overlying, peri-ciliary plasma membrane. The concentrated SAG1 at this ciliary staging area then moves in a mechanism independent of anterograde IFT, into the ciliary membrane. Once in the ciliary membrane, and upon interacting with its cognate receptor SAD1 on the ciliary membrane of the minus gamete, the protein is released into the medium in the form of ciliary ectosomes.

Many elements of this model are in keeping with regulated trafficking of cell surface receptors in other systems. In the canonical mechanism, ligand binding triggers internalization of receptors, which can be targeted to lysosomes for degradation as part of receptor down-regulation or can recycle to reappear at the cell surface [30, 31]. A major element of regulated SAG1 trafficking, however, differs from this canonical pathway in that the SAG1 molecules that are internalized have not bound their ligand, SAD1. Indeed, SAG1 on the plasma membrane of gametes is incapable of binding SAD1, and only when SAG1 reaches the ciliary membrane does it become active [11]. The molecular basis of this inactivity is unknown, but possibly the presence of plasma membrane SAG1 as puncta reflects a hetero-or z organization that prevents ligand binding. Internalization of inactive, non-ligand bound plasma membrane SAG1 triggered by signals generated when active SAG1 on the cilia binds ligand is reminiscent of the so-called non-canonical mechanisms for internalization of “bystander” EGF receptors [32, 33] and of cAMP-regulated internalization of the sodium hydrogen exchanger in vertebrate cells [34].

Internalization and microtubule-associated movement versus lateral transport

Although the mechanism we uncovered in large part is consistent with canonical models for vesicle-based receptor trafficking, several considerations actually supported lateral transport as a feasible mechanism. In the widely studied Hedgehog signaling pathway, mobilization of the effector membrane protein Smoothened from the plasma membrane into the primary cilium has been reported to occur via lateral transport of pre-existing proteins [35] (but see [36] for an alternative model), although ciliary enrichment was observed not to depend on cytoplasmic microtubules [37]. Moreover, several ciliary membrane proteins, including PKD2-like [38] and the ciliary membrane protein FMG1 [39] in Chlamydomonas, have been shown to be transported laterally within the ciliary membrane, almost certainly powered by IFT motors moving along microtubules [40].

The trafficking route identified here, however, overcomes several limitations of a lateral transport mechanism. For example, transmembrane proteins and sub-membranous protein complexes could hinder rapid protein movement [41]. Moreover, a single vesicle transported intracellularly on a microtubule track can carry many copies of a protein the entire length of the track. We should note that the cAMP-triggered redistribution of internalized SAG1-HA in many respects mirrors the microtubule motor-dependent, cAMP-mediated redistribution of pigment granules in melanophores and other pigment-containing cells [42].

Ciliary membrane protein trafficking during ciliogenesis and homeostasis

Mechanisms for regulated entry of pre-existing membrane proteins into fully formed cilia likely incorporate elements related to those functioning during ciliogenesis and during constitutive entry of newly synthesized proteins during ciliary membrane turnover. During ciliogenesis, newly synthesized, cilium-destined membrane proteins are delivered from the Golgi to the base of the organelle by vesicular transport [43–45]. There, the vesicles undergo exocytosis followed by movement of their constituent membrane proteins into the growing organelle through action of IFT-A and Tulp3 [3, 46]. After ciliogenesis, proteins also continue to move into the organelles as a part of constitutive turnover. In photoreceptor rod cells, the photosensory G-protein coupled receptor (GPCR) rhodopsin functions in a modified cilium and is constantly shed at the tip of the rod outer segment [47, 48]. The rhodopsin is replenished by trafficking of vesicles bearing newly synthesized protein from the Golgi directly to the base of the connecting cilium [49].

That Chlamydomonas gametes use this internalization/microtubule-associated transport/externalization mechanism uncovered here for redistribution of pre-existing SAG1 raises the possibility that the mechanism is broadly used. For example, trafficking of the microbial rhodopsin, channelrhodopsin-1, to the eyespot occurs on the rootlet microtubules and depends on IFT, although it is not yet known whether the protein moves laterally within the membrane or in the form of intracellular vesicles [50, 51]. Cells also might use this mechanism to recruit pre-existing membrane proteins to cilia during ciliogenesis. Classic experiments from the 1970’s [52] showed that de-ciliated Chlamydomonas cells possess stores of ciliary proteins sufficient to grow 2 half-length cilia. Vectorial labelling studies showed that the plasma membrane is at least one source for these ciliary membrane proteins, including the agglutinin portion of SAG1 and the ciliary membrane protein FMG1 [11]. Studies in the green alga, Euglena gracilis, showed that pre-existing cell body plasma membrane proteins were incorporated into growing cilia [53]. In future experiments, it will be important to determine whether FMG1 and PKD2 in Chlamydomonas and ciliary membrane proteins in other organisms follow a similar route to the cilium during ciliogenesis.

STAR*Methods

Lead Contact And Materials Availability

Requests for further information or resources and reagents should be directed to and will be fulfilled by the Lead Contact, William J. Snell (wsnell1@umd.edu). This study did not generate new unique reagents.

Experimental Model and Subject Details

Previously described Chlamydomonas reinhardtii mating type plus strain SAG1-HA, which is the sag1–5 mutant strain (CC1146) rescued for ciliary adhesion with plasmid pBSAG1-HA [13], was used for these experiments. The full-length SAG1 protein is cleaved soon after synthesis to yield an N-terminal, aqueous-soluble portion termed the agglutinin, a predicted middle fragment with 3 predicted transmembrane domains, and the HA-tagged C-terminal ~65 kDa fragment with 4 predicted transmembrane domains being analyzed here [13]. Mass spectrometry analysis of SAG1-HA-containing ciliary membrane fractions suggests that the cleaved fragments remain associated with each other (Ranjan and Snell, unpublished). Vegetative cell growth in liquid TAP medium on a 13/11 light-dark cycle with aeration and induction of gametogenesis by transfer into nitrogen-free minimal medium (M-N) and aeration overnight in continuous light were as described previously [13]. Cell numbers were determined by use of a hemocytometer.

Method Details

Gamete activation

To initiate gamete activation, gametes were incubated in medium containing 15 mM dibutyryl cAMP and freshly prepared 0.15 mM papaverine in M-N medium [17].

Protease sensitivity assay

Naive or activated gametes (3×10⁶ −1×10⁷ cells/ml) were harvested from M-N media by centrifugation. For the intact cell samples, portions of the cells were incubated in M-N containing 0.05% trypsin at room temperature (prepared from a 100X trypsin stock solution in 1 mM HCL). After 5 minutes, the cells were harvested by centrifugation and resuspended in M-N containing 1% trypsin inhibitor and 1X protease inhibitor cocktail, washed two times in the same buffer, resuspended in 2X (final concentration) SDS-PAGE sample buffer, and immediately heated at 95°C for 5 minutes in preparation for SDS-PAGE and immunoblotting. For the detergent lysate samples, sedimented cells were quickly frozen in liquid nitrogen. Ice cold 0.1X PBS containing 0.01% Tween-20 was added to the frozen pellets. After thawing, the samples were put through 4 more cycles of freezing and thawing. The final lysates were incubated at room temperature with 0.05% trypsin, prepared from a 100X trypsin stock solution in 1 mM HCL. After 5 minutes, trypsin inhibitor (final concentration 1%), protease inhibitor cocktail (final concentration 1X), and SDS-PAGE sample buffer (final concentration 2X) were added to the lysates and the samples were immediately heated at 95°C for 5 minutes in prepara tion for SDS-PAGE and immunoblotting. In some cases, samples were flash frozen in liquid N2 and stored at −80°C before SDS-PAGE.

SDS-PAGE and Immunoblotting

For immunoblotting, samples were separated by SDS-PAGE on 4–20% SDS-MOPS gradient gels and transferred onto PVDF membranes as described previously [13, 14]. Membranes were blocked by incubation in 3% fat-free dried milk for 1 hour followed by 1 hour of incubation in primary antibody. Membranes were washed three times for 10 minutes with TBST (Tris-buffered saline, 0.1% Tween 20) followed by incubation with secondary antibody and incubation in chemiluminescent substrate. Fluorescence signals were captured on a C-Digit blot scanner (LI-COR Instruments, USA). The antibodies used for immunoblotting were rat anti-HA (1:2000) and goat anti-rat (1:5000); mouse anti-α-tubulin (1:5000) and goat anti-mouse (1:5000); rabbit anti-clathrin (1:3000) and goat anti-rabbit-HRP (1:5000).

Immunostaining, microscopy and image processing

Naive or activated gametes (50–100 μl of 3 × 10⁶ − 1 × 10⁷ cells/ml in M-N) were placed on poly-L-lysine coated-coverslips prepared as previously described [14, 51]. After 10 minutes, 100–200 μl of 3.7% paraformaldehyde in M-N were added to the coverslips in 6-well plates and incubated for 5–10 minutes at room temperature, followed by fixation in 100% methanol at −20°C. To add the methanol (and all subsequent solutions described below), a 1 ml pipette tip containing the liquid was touched to the wall of the well as the solution was expelled. Plates were gently agitated at regular intervals at −20°C. Afte r 20 minutes, the coverslips were incubated in 2–3 ml PBS containing 0.25 M NaCl at room temperature for 10 minutes followed by 5-minute washes with PBS three times and two times with PBS containing 0.5% triton X-100 (PBST). After blocking in 3% goat antiserum in PBS for 1 hour at room temperature, samples were incubated overnight in 1.0 – 1.5 ml primary antiserum or control serum (1:125–1000 dilutions) in PBS at 4°C in a moist chamber. After washing two times with PBST, and once with PBS, the coverslips were incubated with freshly prepared Alexa-Fluor–conjugated secondary antibodies (1: 500–1000 in PBS) for 1 hour at room temperature. Secondary antibody-treated coverslips were washed two times with PBST, three times with PBS, and mounted on slides with anti-fade reagent. Cell visualization and imaging were performed on a Hyd detector-equipped Leica TCS SP5 confocal microscope using a 1.4 numerical aperture, 63 X oil immersion objective). Simultaneous imaging of cells stained for SAG1-HA and tubulin was performed on a Leica TCS SP8 confocal microscope and each z-stack image was processed in real time using the Leica “Lightning” module available in the Leica Application Suite X (LAS X) imaging software (Leica, Germany). Where indicated, images obtained from a z-series were summed to produce a projected image using Leica LAS X or ImageJ software (NIH, USA), and cropped in the Illustrator program of Adobe Systems Inc. (USA).

Quantification and Statistical Analysis

Results shown are typical of experiments performed three to five times. For immunolocalization, at least 200 cells were observed in each experiment.

Data And Code Availability

This study did not generate code and did not analyze datasets or code.

Supplementary Material

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Video S1. Z-stacks of the longitudinal view of the cell immuno-stained for SAG1-HA. The maximum intensity projection is in Figure 3A, lower panel. Related to Figure 3A.

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Video S2. Z-stacks of the cross-sectional view of the cell immuno-stained for SAG1-HA. The maximum intensity projection is shown in Figure 3B. Related to Figure 3B.

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Video S3. Z-stacks of the longitudinal section of the cell immuno-stained for tubulin. The maximum intensity projection is shown in Figure 4A-i. Related to Figure 4A-i.

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Video S4. Z-stacks of the cross-sectional section of the cell immuno-stained for tubulin. Three individual Z-stacks are shown in Figure 4A-ii. Related to Figure 4A-ii.

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Video S5. Z-stacks of the cross-sectional view of the cell doubly immuno-stained for SAG1-HA and tubulin. The maximum intensity projection is shown in Figure 4C. Related to Figure 4C.

Highlights.

• During signaling, a plasma membrane pool of ciliary receptors is internalized

• In transit to cilia, internalized receptors align along cytoplasmic microtubules

• Within minutes after internalization, receptors polarize to the bases of the cilia

• Receptors re-appear on the cell surface as they enter the ciliary membrane