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- Article: found
Non-Overlapping Distributions and Functions of the VDAC Family in Ciliogenesis
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Abstract
Centrosomes are major microtubule-organizing centers of animal cells that consist of two centrioles. In mitotic cells, centrosomes are duplicated to serve as the poles of the mitotic spindle, while in quiescent cells, centrosomes move to the apical membrane where the oldest centriole is transformed into a basal body to assemble a primary cilium. We recently showed that mitochondrial outer membrane porin VDAC3 localizes to centrosomes where it negatively regulates ciliogenesis. We show here that the other two family members, VDAC1 and VDAC2, best known for their function in mitochondrial bioenergetics, are also found at centrosomes. Like VDAC3, centrosomal VDAC1 is predominantly localized to the mother centriole, while VDAC2 localizes to centriolar satellites in a microtubule-dependent manner. Down-regulation of VDAC1 leads to inappropriate ciliogenesis, while its overexpression suppresses cilia formation, suggesting that VDAC1 and VDAC3 both negatively regulate ciliogenesis. However, this negative effect on ciliogenesis is not shared by VDAC2, which instead appears to promote maturation of primary cilia. Moreover, because overexpression of VDAC3 cannot compensate for depletion of VDAC1, our data suggest that while the entire VDAC family localizes to centrosomes, they have non-redundant functions in cilogenesis.
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Cep164, a novel centriole appendage protein required for primary cilium formation
Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. Through a small interfering RNA screen we found several centrosomal proteins (Ceps) to be involved in PC formation. One newly identified protein, Cep164, was indispensable for PC formation and hence characterized in detail. By immunogold electron microscopy, Cep164 could be localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These data implicate distal appendages in PC formation and identify Cep164 as an excellent marker for these structures.
- Record: found
- Abstract: found
- Article: found
MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis
Corey Williams, Chunmei Li, Katarzyna Kida … (2011)
Introduction Primary cilia protrude from most mammalian cells and modulate sensory processes, including chemo-, mechano-, and photo-reception (Fliegauf et al., 2007). Cilia regulate various signaling pathways during embryonic development and are needed for normal postnatal tissue homeostasis (Gerdes et al., 2009). Mutations disrupting ciliary functions cause human disorders (ciliopathies) that collectively affect nearly all tissues/organs (Sharma et al., 2008). A nonexhaustive list of ciliopathies includes Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS), Senior-Løken syndrome (SLSN), Leber congenital amaurosis (LCA), polycystic kidney disease (PKD), and oral-facial-digital syndrome (OFD). These disorders present with variable but overlapping clinical phenotypes that encompass polycystic kidneys, liver fibrosis, skeletal anomalies, sensory impairment, and brain/nervous system deformities (Fliegauf et al., 2007). At least 35 loci have been identified in ciliopathy patients, some of which contribute to multiple seemingly distinct syndromes (Baker and Beales, 2009). Many of these genes encode proteins that localize to the basal body (BB)—a centriolar structure universally required for extending the microtubule-based ciliary axoneme—or to an adjacent domain, termed “transition zone” (TZ) in most cilia, or “connecting cilium” in photoreceptors (Horst et al., 1990; see schematic of BB-TZ-cilia structures in Fig. 1 A and relevant disease proteins in Table S1 A). Within the BB-TZ region are subdomains that include transitional fibers (TFs) and Y-links. TFs form a pinwheel-like structure, of unknown protein composition, that links the BB to the proximal ciliary membrane. The Y-links of the TZ connect—via high-affinity linkages—axonemal microtubules to the membrane at the ciliary necklace, a proteinaceous decoration of the TZ membrane (Muresan and Besharse, 1994). Together, the TFs and TZ are proposed to form a gate (Rosenbaum and Witman, 2002; Satir and Christensen, 2007) that excludes vesicles from cilia, prevents unwanted diffusion of membrane proteins into cilia, and selectively regulates protein ciliary entry and exit (Fig. 1 A). Figure 1. C. elegans B9 and C2 domain ciliopathy proteins are found at the transition zone, adjacent to the basal body/transition fiber region. (A) Schematic of a prototypical basal body (BB)/transition zone (TZ)/cilium, highlighting the microtubule (MT) backbone of the organelle, the docking of vesicles at the base of a “ciliary gate,” and its intraflagellar transport (IFT) trafficking machinery. In C. elegans, the BB lacks discernable MTs (shown dashed) and consists mainly of transition fibers (TFs). The TZ is demarcated by Y-links spanning the axoneme to the membrane and likely organizes the “ciliary necklace” on the ciliary membrane. The TFs and TZ are proposed to form a gate that restricts entry of vesicles and potentially also nonciliary proteins. TEM cross sections from C. elegans show relevant substructures (TFs, TZ, middle and distal cilia segments; Bars, 100 nm). IFT particles carry cargo from the BB into cilia; kinesins transport two multi-protein complexes (IFT subcomplexes A and B) and a BBS protein complex (S) along with cargo, and dynein recycles components back to the BB. (B) MKS-1::YFP localization to the TZ in relation to the CHE-13 IFT protein, which concentrates at BB/TFs and is present along the axoneme. Two phasmid (tail neuron) cilia are shown, as in C and D; all tagged proteins are expressed under endogenous promoters unless specified otherwise; MS, middle segment; DS, distal segment. Bar, 2 µm. (C and D) Similar to MKS-1, MKS-5::tdTomato and MKS-6::GFP (both osm-5 promoter driven) localize to the TZ, which largely does not overlap with the peak intensities of tagged IFT proteins (DYF-11 and XBX-1, respectively) at the adjacent BB/TFs. Bars, 2 µm. (E) B9 domains of MKS-1, MKSR-1, and MKSR-2 may be structurally related to C2 domains of RGRIP1L/MKS-5 and CC2D2A/MKS-6 (see F and G). A representative structure of synaptotagmin I C2 domain (PDB code 1byn), with bound Ca2+, is shown. (F) A Hidden Markov Model profile was created using B9 domains from C. elegans, C. briggsae, and C. remanei to search the C. elegans proteome for related domains in evolutionarily conserved proteins. Only four are retrieved: the B9 input proteins and a C2 domain protein, synaptotagmin-4 (SNT-4). (G) The top hits from the structure prediction algorithm GenTHREADER reveal that all three human B9 domains can be modeled onto known C2 domain NMR/crystal structures from different proteins (synaptotagmins, E3 ubiquitin-protein ligase NEDD4-like, phospholipase C-delta1, and rabphilin-3A; PDB codes in parentheses). Axoneme elongation is thought to initiate when the mother centriole docks with a membrane either at the cell surface or a ciliary vesicle in the cytosol (see Fig. 10 D; Sorokin, 1962). Full extension of the axoneme then relies on an intraflagellar transport (IFT) machinery that uses kinesin and dynein motors and associated subcomplexes (IFT-A, IFT-B, and BBSome) to traffic ciliary cargo from TFs to the cilium tip and back (Fig. 1 A; Silverman and Leroux, 2009). In contrast to the extensive characterization of IFT-mediated axoneme extension, the components and mechanism involved in BB/TZ membrane association and establishment of the ciliary gate remain virtually unknown. Based on knockdown studies in mammalian cells, the ciliopathy proteins MKS1 and MKS3 have been implicated in BB migration/docking and thus ciliogenesis (Dawe et al., 2007). However, these defects are absent from rodent Mks1 or Mks3 mutants (Tammachote et al., 2009); thus, the role of these and most other BB/TZ-associated ciliopathy proteins remains unclear. Recently, the ciliopathy protein CEP290 was localized to TZ Y-links, and its disruption in Chlamydomonas altered the ciliary composition of IFT components and other proteins (Craige et al., 2010); Caenorhabditis elegans NPHP-1 and NPHP-4 have also been proposed to act in ciliary gating (Jauregui et al., 2008). Whether additional TZ or IFT proteins are similarly involved in regulating ciliary gating, and the mechanism by which they perform these functions, is not known. Here, we used C. elegans to elucidate the functions of eight conserved proteins, six of which are MKS/NPHP associated. We find that MKS-5/RPGRIP1L interacts with two distinct TZ functional modules, MKS/MKSR and NPHP, consisting of MKS-1/MKSR-1/MKSR-2/MKS-3/MKS-6 and NPHP-1/NPHP-4 proteins, respectively. Functional interactions between different MKS module components and the NPHP module are essential for an IFT-independent early stage of ciliogenesis, namely docking/anchoring of the BB/TZ to the membrane. Moreover, the two modules restrict inappropriate accumulation of membrane-associated proteins inside cilia. Our findings help to comprehensively define an interaction network of ciliopathy-associated proteins and allow us to propose for the first time a unified model for the function of diverse MKS/NPHP proteins, in which the MKS and NPHP modules altogether enable associations between microtubules and the ciliary membrane; this ciliogenic event coincides with construction of the ciliary gate that establishes the specialized compartment. Results MKS/MKSR and NPHP proteins localize specifically to the ciliary TZ To gain insights into MKS/MKSR/NPHP protein functions, we first defined their respective subcellular localization in C. elegans sensory neurons. Using fluorescently tagged proteins, we detect MKS/MKSR/NPHP proteins in a region corresponding to the TZ (adjacent to where IFT proteins concentrate at the TFs/BB). This is evident for MKS-1, MKS-1 related-1 (MKSR-1)/B9D1, MKS-1 related-2 (MKSR-2)/B9D2, MKS-3/meckelin, NPHP-1, and NPHP-4 (Winkelbauer et al., 2005; Williams et al., 2008, 2010; Bialas et al., 2009), as well as MKS-5/RPGRIP1L and MKS-6/CC2D2A, previously uncharacterized in C. elegans (Fig. 1, B–D; Fig. 2). By transmission electron microscopy (TEM), the ∼0.8-µm long TZ region containing Y-links is distal to the TFs, which sit at the ciliary base just inside the dendritic tip (Fig. 1 A; Perkins et al., 1986). These data suggest that the TZ represents a common site of dysfunction in MKS/NPHP ciliopathy patients; however, the functions of MKS/MKSR/NPHP proteins at the TZ remain undetermined. Notably, based on Hidden Markov Model profiling and structure/fold predictions, several TZ proteins (MKS-1, MKSR-1, MKSR-2, MKS-5, and MKS-6) share a related C2/B9 motif (Fig. 1, E–G; Table S1 C). This motif is predicted to bind Ca2+/lipids and participate—similar to synaptotagmin—in membrane/vesicle trafficking and fusion (Nalefski and Falke, 1996). The presence of this motif in multiple-ciliopathy TZ proteins raises the possibility that they perform a shared function at the TZ. Figure 2. MKS/MKSR and NPHP proteins localize at the ciliary TZ. (A) Fluorophore-tagged MKS/MKSR and NPHP proteins localize at the TZ in tail cilia. Each TZ protein signal is overlaid with IFT or other TZ protein signal, as indicated. Bar, 10 µm. (B) MKS-5::tdTomato and MKS-6::GFP localize at the TZ in head cilia. MKS-5 and MKS-6 are overlaid with IFT proteins DYF-11::GFP and XBX-1::tdTomato, respectively. Bar, 10 µm. (C) GFP-tagged MKS-1, MKSR-1, and MKS-6 localize at the base of ciliary axonemes (consistent with the TZ), which are marked by staining with an antibody against polyglutamylated tubulin. Bar, 10 µm. (D) Schematic of MKS/MKSR/NPHP protein localization at the TZ. Functional interactions between NPHP proteins and MKS-5 or MKS-6 are required for ciliogenesis To examine potential ciliary roles of the uncharacterized C2 domain–containing MKS-5 and MKS-6 proteins, we first analyzed mks-5 and mks-6 mutants (Fig. 3 A). Similar to mutations disrupting B9 domain genes (mks-1, mksr-1, and mksr-2; Williams et al., 2008; Bialas et al., 2009), mks-6(gk674) mutants have no overt cilia or IFT defects, based on: (1) normal uptake of hydrophobic fluorescent dye (DiI), indicating cilia are present and environmentally exposed (Fig. 3, B and D); (2) normal cilia length, observed by visualizing cilia with GFP-tagged IFT proteins (Fig. 4, B and C; Fig. 5, A, A′, B, and B′); and finally, (3) normal IFT rates, based on in vivo time-lapse microscopy (Table S1 D). TEM analysis verifies the absence of gross cilia ultrastructure anomalies in mks-6(gk674) mutants, except for occasional loss of axoneme distal segments (Fig. 6). mks-6(gk674) mutants have a weak osmotic avoidance phenotype comparable to nphp-4(tm925) mutants (Fig. 4 D) but do not exhibit increased lifespan, unlike other cilia gene (e.g., IFT) mutants (Table S1 E). Analysis of mks-5(tm3100) mutants uncovered moderate defects in dye filling and phasmid (tail) cilia length (Fig. 3, C and D); however, this was not accompanied by prominent changes in either osmotic avoidance, distribution of IFT proteins to BBs/cilia (Fig. 4, A and C; Fig. 5, A, A′, B, and B′), or IFT rates (Table S1 D). Figure 3. Functional interactions between MKS-5/MKS-6 and other TZ proteins are required for environmental exposure of cilia to a fluorescent dye, consistent with a role in ciliogenesis. (A) Gene structures of C. elegans mks-5 (C09G5.8) and mks-6 (K07G5.3) and the nature of three deletion alleles, tm3100, gk674, and nx105. (B and C) Representative images of fluorescence staining of environmentally exposed sensory neurons via DiI uptake through cilia in head (amphid) neurons (left panels) and tail (phasmid) neurons (right panels). Dye filling is noted as normal (+++), reduced (+), or absent (−). (B) Wild-type (N2) and mks-6 strains exhibit normal dye filling; nphp-1 and nphp-4 show normal or slightly reduced (++) dye filling (see D; Jauregui et al., 2008). mks-6;mks-5, mks-6;nphp-1, and mks-6;nphp-4 double-mutant combinations show little or no dye filling. Expressing GFP-tagged MKS-6 rescues the mks-6 (double) mutant phenotype. (C) mks-5(tm3100) mutants have reduced dye filling, whereas mks-5;mksr2, mks-5;mks-6, and mks-5;nphp-4 double mutants show little to no dye filling. Bars, 17.5 µm. (D) Summary of dye-filling phenotypes in single and double mks/mksr/nphp mutants. NT, not tested; red, present study; black, data from Jauregui et al. (2008) and Williams et al. (2008, 2010). Figure 4. Functional interactions between TZ proteins are required for correct cilium length and positioning of basal bodies/TZs, as well as chemosensation. (A and B) Disruption of mks-5 (A) or mks-6 (B) has no major effect on the gross morphology/presence of amphid (head) and phasmid (tail) cilia, as revealed by GFP-tagged IFT markers (OSM-6 and CHE-2, respectively). BB, basal body. Bars, 5 µm. (C) Analyses of amphid and phasmid cilia length (white and light gray bars), spatial distribution of BB/TZ in amphid channel neurons (black bars), and position of the BB with respect to the cell body in tail neurons (dark gray bars) are summarized based on phenotypes and statistical analyses shown in Fig. 5. Mean values obtained for wild-type (N2) are shown; *, statistically significant difference (P 40) of the measurements in A–D are shown with a bar graph showing average and standard error. Statistically significant differences (P < 0.01) in the mean (*) or variance (§) are indicated for all combinations. Figure 6. MKS-6 and NPHP-4 are collectively required for BB/TZ attachments to membrane. Shown are low and high magnification images from TEM serial cross sections of amphid channel cilia from wild-type (N2), mks-6, and mks-6;nphp-4 worms. Top row, distal segments (DS); middle row, middle segments (MS); bottom row, transition zone (TZ) and distal dendrites (DD). Below bottom row are magnified images of representative TZs. Boxed number denotes proximal positioning of section relative to top section. Schematics (longitudinal, transverse views) summarize the major ultrastructural observations. BB, basal body; TFs, transition fibers. Bars, 100 nm. (A) N2 worms showing 10 singlet microtubule (MT)-containing axonemes in DS, 10 doublets in MS (+3 µm), and TZs (constriction of MT doublets surrounding apical ring) with intact Y-links (connecting doublets with ciliary membrane) anchored at distal dendrite tips (+7 µm). TFs are also observed below this region. (B) Apart from rare axonemes lacking MTs in DS, mks-6 mutant cilia possess normal DS, MS (+2 µm), and TZ (+5 µm) regions. (C) In DS and MS (+4 µm) regions of mks-6;nphp-4 worms, many axonemes are missing and amphid pore size is reduced. Open/unzipped B-tubules are also observed, as in nphp-4 mutants (Jauregui et al., 2008). In TZ/DD region, TZs are not anchored at the membrane of the DD tips, but instead are often mispositioned in more proximal regions of the dendrites (see also schematic). Y-links at the TZ are missing and TFs are not observed. Previously, we found that B9 domain proteins (of the MKS/MKSR module) functionally interact with NPHP-1 and NPHP-4 (NPHP module) to facilitate ciliogenesis (Williams et al., 2008). To determine if similar genetic interactions occur between MKS-5 and MKS-6 and the MKS/MKSR or NPHP modules, we examined mks-5 and mks-6 mutations in combination with mks/mksr and nphp mutations. Compared with wild-type, mks-5, mks-6, or nphp-4 single mutants, both mks-5;nphp-4 and mks-6;nphp-4 double mutants exhibit strong dye-filling (Dyf) and osmotic avoidance (Osm) phenotypes (Fig. 3, B–D; Fig. 4 D). However, dye filling is normal in mks-6;mks-1, mks-6;mksr-1, mks-6;mks-3, and mks-6;mksr-2 double mutants (Fig. 3, B and D). These data suggest that mks-6 genetically interacts with the NPHP module and is associated with the MKS/MKSR module. Interestingly, combining mks-5 mutants with mksr-1, mksr-2, or mks-6 mutations also results in a stronger Dyf phenotype (Fig. 3, C and D), suggesting that mks-5 genetically associates with not only the NPHP module but also the MKS/MKSR module. To study in greater detail the ciliary defects of single and double mks/mksr/nphp mutants, we used GFP-tagged ciliary (IFT) markers. We uncovered in double mutants multiple phenotypes, including missing or shorter cilia (Fig. 4 C; Fig. 5, A, A′, B, and B′), misoriented cilia (not depicted), and improperly positioned BB/TF regions within the amphid channel and with respect to the phasmid cell body (Fig. 4 C; Fig. 5, C, C′, D, and D′; Williams et al., 2008). Although there is a low penetrance of these phenotypes in nphp-4 single mutants, they were consistently observed in mks-6;nphp-4, mks-5;nphp-4, and mks-6;nphp-1 double mutants (Fig. 4 C; Fig. 5). Joint disruption of an MKS/MKSR protein and NPHP-4 results in BB/TZ membrane association defects To assess the nature of the ciliary ultrastructural defects in mks/mksr/nphp double mutants, we used TEM. Strikingly, we observed profound and largely indistinguishable phenotypes across all double mutants analyzed. Unlike wild-type animals and mks-6 or nphp-4 single mutants, which show normal TZs (Fig. 6, A and B; Fig. 7; Jauregui et al., 2008), mks-6;nphp-4 and mks-5;nphp-4 double mutants exhibit mispositioned and disrupted TZ regions, with clear disconnections between the BB/TZ region and membrane, accompanied by missing Y-links (Fig. 6 C; Fig. 7; Fig. S1, Fig. S2); hence, their BB/TZ regions are not properly anchored at the distal end of the dendritic membrane. We also observed missing, misplaced, or shorter axonemes, and variably missing TFs. In perhaps the most severe instance of morphological abnormality, mks-5;nphp-4 double mutants had incomplete TZ microtubule rings (Fig. S2). Phenotypes nearly identical to mks-6;nphp-4 mutants were observed in the mksr-1;nphp-4 strain (Fig. 7; Fig. S3). The various cilia defects uncovered by TEM are quantified in Table I. Notably, all TZ/cilia anomalies are in addition to open tubules observed in the nphp-4 mutant (Jauregui et al., 2008) and are distinct from those of IFT and other cilia mutants, in which TZ regions are intact (Perkins et al., 1986). Table I. Quantification of various ciliary ultrastructure defects in TZ protein–disrupted strains Region Feature/defect N2 mks-6 mks-6; nphp-4 mksr-1; nphp-4 mks-1; nphp-4 mks-5; nphp-4 mks-3; nphp-4 DSa Ave. axoneme no./pore (n = 2–4) 10 10 3 2 6 5 5 Ave. MT singlet no./axoneme (n = 3–44) 8 7 5 7 8 4 6 No. of axonemes with no MTsd 0 4 9 3 8 3 2 MSb Ave. axoneme no./pore (n = 2–4) 12 12 5 4 11 9 7 Ave. MT doublet no./axoneme (n = 12–100) 9 8.9 7.7 6.0 8.3 8.0 7.5 Axonemes with abnormal accumulationsd e 0 0 10 0 0 2 0 TZc Ave. diameter of surrounding membrane (nm) (n′ = 2–34) 260 262 564 436 286 337 320 Percentage with enlarged membrane diameters (n′ = 2–34) 0 0 100 100 25 100 60 Percentage of TZs with Y-links (n′ = 2–14) 100 100 0 0 50 0 30 DD Abnormal vesicle or membrane accumulationsd e 0 0 23 4 2 5 4 DS, distal segment; MS, middle segment; TZ, transition zone; DD, distal dendrite; N, number of amphid pores analyzed; n, number of axonemes measured; n′, number of TZs analyzed. a Singlet microtubule (MT) axoneme region. b Doublet MT axoneme region. c Drawing together of doublet and singlet MTs by the apical membrane; doublets linked to ciliary membrane via Y-links. d As analyses were performed on serial sections, the same axoneme may have been counted multiple times. e Electron-dense accumulations, often vesicular (30–60 nm; often tubular; clear or dense core), sometimes non vesicular (<20 nm). Figure 7. Functional interactions between five TZ-associated proteins (MKS-1, MKSR-1, MKS-3, MKS-5, and MKS-6) and NPHP-4 contribute to anchoring the BB/TZ to membrane. (A) TEM images of amphid channel TZ regions obtained from longitudinal sections of wild-type (N2) and mks-6;nphp-4 double mutants. Compared with N2 worms in which membrane (green outlines) associates tightly with the TZ (demarcated by dashed lines), mks-6;nphp-4 double-mutant TZs fail to anchor to the surrounding membrane (red outlines). Bars, 500 nm; 200 nm (insets). (B) TEM images of amphid channel ciliary TZ regions obtained from cross sections of mks-3;nphp-4, mks-5;nphp-4, mks-1;nphp-4, mksr-1;nphp-4 and mks-6;nphp-4 double mutants. Boxed number denotes the proximal positioning of the imaged section relative to the most distal sections of the amphid pore. In all worms, the MT doublet-containing TZ ring is not anchored at distal dendrite tips (which are typically positioned at +5 to +6 µm relative to distal amphid pore); instead, TZs are found at abnormal proximal positions in the dendrites (+8 to +16 µm). Y-links are frequently not observed. Bars, 100 nm. Given the severe ciliary phenotypes of mks-5;nphp-4 and mks-6;nphp-4 strains, we wondered if mks-1;nphp-4 and mks-3;nphp-4 mutants, which have milder Dyf phenotypes (Williams et al., 2008, 2010), would present with similar—yet partial or more specific—ultrastructure defects. Although similar ciliary defects were observed in the latter mutants (Fig. 7; Fig. S4, Fig. S5), some of their TZ regions are in normal proximity to the ciliary membrane but often lack connecting Y-links; this raises the possibility that MKS/MKSR/NPHP proteins comprise components of these structures or are required for their stability. TZ proteins participate in early ciliogenesis independent of IFT-associated proteins Our data indicate that MKS/MKSR/NPHP proteins are important for BB/TZ interactions with the membrane, which likely occur during ciliogenesis before IFT-dependent axoneme extension, suggesting a role in an early step of the ciliogenic pathway (Sorokin, 1968). To test if this early step depends on IFT, we looked for IFT defects using kymograph analyses in mks/mksr/nphp double mutants where a small proportion of cilia still form. Remarkably, IFT was largely unaffected in these double (as well as single) mutants (Table S1 D; Bialas et al., 2009), in striking contrast to C. elegans IFT mutants where disruption of anterograde or retrograde IFT occurs (Hao and Scholey, 2009), or BBS mutants (bbs-1/7/8), where stability/association of IFT subcomplexes A and B is compromised (Blacque et al., 2004; Ou et al., 2007). Because no TZ phenotypes observed in our study are apparent in IFT or BBS mutants (Perkins et al., 1986; unpublished data), and because IFT is largely unaffected, we propose that NPHP and MKS protein modules act before IFT-driven axoneme formation. From this model we predict that the TZ should be established properly when IFT is disrupted. This was validated in IFT and BBS mutants, where MKS-6 (Fig. 8 F) and other MKS/NPHP proteins (unpublished data) localize normally to the TZs of cilia lacking fully formed axonemes. Together, these data support a model where TZ proteins function in an early ciliogenic step (BB/TZ association with membrane) followed by a subsequent IFT/BBS-dependent step in axoneme elongation/trafficking. Figure 8. MKS/MKSR and NPHP proteins are organized hierarchically as modules, and independently of IFT/BBS proteins, at the TZ. (A) Schematic representing the region of phasmid (tail) cilia analyzed in B–E. Entire cilia/cilia regions are shown in F–K. (B–E) Co-dependent localization of fluorophore-tagged TZ proteins. TZs lacking normal localization of a particular protein are circled. (B) MKS-6::GFP localizes normally in wild-type (N2) and mks-1, mks-3, and nphp-4 mutants, but mislocalizes in mks-5, mksr-1, and mksr-2 mutants. (C) mks-5 mutants fail to localize MKS-1, MKSR-1, MKSR-2, and MKS-3, the latter dispersing along the cilium axoneme (see also Fig. 9 L). NPHP-1 and NPHP-4 localize to the TZ in mks-5 mutants, but to a subregion smaller than that occupied in N2 (compare brackets). (D) MKS-5::tdtomato localization at the TZ is not perturbed in mksr-2, nphp-4, or mks-6;nphp-4 double mutants. (E) MKS-3::GFP localizes correctly to the TZ in nphp-4 and mks-6 single mutants but is mislocalized in the double mutant. (F) MKS-6::GFP localizes normally in bbs mutants (bbs-7, bbs-8) and ift mutants (osm-5, che-11), indicating the presence of an intact TZ. (G) tdTomato-tagged NPHP-1 (top) and MKSR-1 (bottom) localize normally at the TZ in mks-6(gk674) mutants. (H–K) CFP- or YFP-tagged MKS-1, MKSR-2, NPHP-1, and NPHP-4 localize normally at the TZ in mks-3 mutants. Bars, 2.5 µm. MKS-5 is a central component required for docking/anchoring MKS and NPHP protein modules Our TEM analyses provide fundamental insights into defects caused when TZ proteins (MKS-1, MKSR-1, MKS-3, MKS-5, and MKS-6) are disrupted jointly with NPHP-4 (Figs. 6 and 7; and Figs. S1–S5). The ultrastructural defects overlap greatly between mutants, implying a shared function for MKS/MKSR/NPHP proteins. Further support comes from biochemical data linking many ciliopathy proteins in shared macromolecular complexes (Table S1 A). To provide further evidence for shared/modular functions, we queried whether disrupting particular TZ proteins affected the localization of others. Using this approach, we rule out MKS-1, MKS-3, MKS-6, and NPHP-1 as critical docking proteins, as their disruption had no effect on localization of other TZ proteins (Fig. 8, B–F; Winkelbauer et al., 2005; Williams et al., 2008, 2010). In contrast, disrupting MKSR-1, MKSR-2, or MKS-5 results in TZ delocalization of MKS-6 (Fig. 8 B) and MKS-3 (Fig. 8 C, Fig. 9 L). Furthermore, mks-5 mutants failed to properly localize MKS-1, MKSR-1, and MKSR-2, suggesting a key role of MKS-5 in docking proteins at the TZ (Fig. 8 C). Notably, NPHP-1 and NPHP-4 localization was also altered in mks-5 mutants (Fig. 8 C), albeit differently; instead of failing to anchor at the TZ, NPHP-1 and NPHP-4 occupied a smaller region than wild-type animals (TZ length of 0.65 µm, n = 19 in mks-5 mutants vs. 1.05 µm, n = 23 in controls; t test P < 0.0001). MKS-5 was unaffected upon disruption of MKSR-2 or NPHP-4, which are otherwise required for TZ docking most other MKS/MKSR proteins and NPHP-1, respectively (Fig. 8 D; Winkelbauer et al., 2005; Williams et al., 2008). Moreover, MKS-5 is still TZ localized in mks-6;nphp-4 double mutants in which ciliary microtubule–membrane attachments are disrupted (compare Fig. 8 D with Fig. 8 E, in which transmembrane MKS-3 is predictably lost in the same double mutant). Thus, we conclude that MKS-5 localizes to the TZ independently of other TZ proteins tested and performs a central role as a scaffold for anchoring other MKS/MKSR and NPHP module proteins. Figure 9. MKS/NPHP proteins are required for ciliary gate function but not for ODR-10 trafficking. (A and B) GFP-tagged ODR-10 odorant receptor concentrates specifically at the AWB cilium in wild-type (N2) animals, showing a typical branched ciliary structure. Bars: (A) 25 µm; (B–K) 8 µm. (C–G) Disruption of the indicated TZ genes does not overtly affect the structure of the AWB cilium or presence of ODR-10::GFP. (H–K) Various double TZ mutants reveal abnormal AWB ciliary structures but otherwise normal localization of ODR-10::GFP to the ciliary membrane. (L) MKS-3::GFP localizes normally at the TZ in nphp-1 and mks-1 mutants but accumulates abnormally inside cilia (cil) and at dendritic tips (DT) in mks-5, mksr-1, and mksr-2 mutants. Bar, 2.5 µm. (M) GFP-tagged RPI-2 is found at dendritic tips but not inside cilia, marked by XBX-1::tdTomato in wild-type worms. RPI-2 localizes normally in mks-1, mks-3, and nphp-1 single mutants but accumulates within cilia of mks-3;mks-1 double mutants and mksr-1, mksr-2, mks-5, mks-6, and nphp-4 single mutants. Bar, 2.5 µm. (N) tdTomato-tagged TRAM-1a is found at dendritic tips but not inside cilia (ciliary TZ marked by MKS-3::GFP) in wild-type worms. TRAM-1a localizes normally in mks-1 and mks-3 mutants but accumulates within cilia of mksr-1, mksr-2, mks-5, mks-6, nphp-1, and nphp-4 mutants. Bar, 2.5 µm. TZ proteins are not required for trafficking of a cilium-targeted protein Given the presence of C2 domains in MKS-5 and MKS-6 and related B9 domains in MKS-1, MKSR-1, and MKSR-2, we hypothesized that these proteins may be required for vesicle trafficking and/or docking/fusion of vesicles harboring ciliary cargo at the TZ (Nalefski and Falke, 1996). Thus, we used a well-established odorant receptor (ODR-10; Sengupta et al., 1996) as a functional marker for vesicular trafficking to cilia. In wild-type animals, ODR-10 (expressed under the str-1 promoter) concentrates at the branched AWB cilium membrane (Fig. 9, A and B). In single TZ mutants, no apparent defects in cilia structure or ODR-10 localization are seen (Fig. 9, C–G). Although AWB cilium morphology in TZ double mutants is compromised, ODR-10 localization to its ciliary membrane appears normal (Fig. 9, H–K). These findings suggest that TZ proteins do not play an essential role in ciliary cargo-associated vesicle trafficking to, and docking/fusion steps at the base of cilia. TZ proteins establish a gate that modulates ciliary composition Our analyses uncovered important roles for MKSR-1, MKSR-2, and MKS-5 in TZ localization of the transmembrane protein MKS-3 (Fig. 8 C; Fig. 9 L). Interestingly, in addition to being lost from the TZ in mksr-1, mksr-2, and mks-5 mutants, MKS-3 accumulates along ciliary axonemes (and at nearby dendritic tips; Fig. 9 L). This indicates that MKSR-1, MKSR-2, and MKS-5 restrict MKS-3 to the TZ membrane. Based on these observations, we wondered if other membrane-associated proteins (in particular, non-TZ-associated proteins) also accumulate in cilia when TZ proteins are disrupted. We examined RPI-2, the C. elegans orthologue of human X-linked retinitis pigmentosa 2 (RP2; Schwahn et al., 1998; Chapple et al., 2000). In wild-type C. elegans, GFP-tagged RPI-2 associates with membrane in sensory neurons, enriched as a ring-like pattern near or at BB/TFs (Blacque et al., 2005), as in trypanosomes (Stephan et al., 2007), but not within cilia (Fig. 9 M). In contrast, we observed that in TZ mutants, RPI-2 accumulates inside cilia (Fig. 9 M). We acquired almost identical results with the transmembrane protein TRAM-1a (Fig. 9 N), which in wild-type animals is found at dendritic tips and excluded from cilia (Bae et al., 2006). Together, our data showing abnormal accumulations of MKS-3, RPI-2, and TRAM-1a in TZ mutant cilia indicate that, in addition to their role in early ciliogenesis, most if not all TZ proteins normally function to maintain a boundary at the cilium base, establishing the TZ as a bona fide ciliary gate. Discussion Modular function reduces the perceived complexity of heterogeneous disorders There are at least 35 genes associated with primary cilia disorders (Baker and Beales, 2009). Several, including BBS genes, are linked to IFT (Blacque and Leroux, 2006; Beales et al., 2007), but most lack clearly assigned molecular functions. Many of these components display genetic and/or physical interactions and are associated with overlapping clinical ailments, suggesting involvement in a common cellular process. Our findings indicate that C. elegans proteins implicated in many ciliopathies, including MKS, NPHP, JBTS, and LCA, function at the TZ and are required for BB and TZ attachment to the membrane and establishing a ciliary gate early in ciliogenesis (Fig. 10 D). Assignment of individual proteins to a particular TZ module (MKS/MKSR or NPHP) simplifies our understanding of the associated ciliopathies. Although previously seen as diseases of complex and seemingly unrelated molecular etiology, our data indicate that MKS and NPHP are likely disorders of macromolecular complexes sharing a common biological function. This is similar to the finding that several BBS proteins are constituents of the multimeric BBSome, which functions as a ciliary coat complex (Nachury et al., 2007; Jin et al., 2010). Indeed, a collective function of TZ proteins is supported by fragmented but increasing biochemical data in mammalian systems (Fig. 10 A; Table S1 A). Other functional modules/complexes may have disparate yet essential ciliary functions that are associated with equivalent ciliopathies; one likely includes NPHP2/inversin, NPHP3, and NEK8, which localize at the Inv ciliary compartment just distal to the TZ (Shiba et al., 2010). Figure 10. MKS/MKSR and NPHP proteins form part of a functional interaction network required for an early stage of ciliogenesis and formation of an intact ciliary gate. (A) Previously identified physical interactions between C. elegans or mammalian TZ proteins (see also Table S1 A). Interactions between MKS1 and MKS3 and between NPHP1, NPHP4, and MKS5 were identified in mammals. The interaction between MKSR1 and MKSR2 was identified in C. elegans. (B) Summary of hierarchy analysis uncovering the requirement of some TZ proteins for normal localization of others. Black arrows represent previously known requirements (arrows point away from the protein required for localization of the other). Red arrows represent novel requirements uncovered in this study. (C) Summary of functional (genetic) interactions between C. elegans TZ components influencing ciliogenesis. Black lines represent previously known functional interactions, and red lines represent genetic interactions uncovered in this study (dye-filling/ciliogenesis and/or BB docking/anchoring and ciliary structures, as revealed by TEM). Modular assignments based on these functional interactions and hierarchy analysis data are represented by stars. (D) Model depicting early and late events in ciliogenesis, first involving transition zone (TZ) proteins and then IFT-associated proteins. Top pathway: in some mammalian cell types, the earliest step of ciliogenesis involves binding of a ciliary vesicle (CV) to the mother centriole, followed by migration of the centriole-CV toward the plasma membrane (PM). At some point a ciliary bud (likely to be a maturing TZ) emerges, invaginating the CV. Bottom pathway: in other cells, the CV may be expendable, and docking of the centriole/developing TZ occurs directly with the PM. During these early steps, interactions between the BB and TZ with the membrane depend on MKS/MKSR/NPHP TZ proteins. Assembly of the TZ in early ciliogenesis likely coincides with formation (of at least part) of the ciliary gate, and MKS/MKSR/NPHP proteins are functional/structural elements of this mechanism. IFT and BBS proteins are not necessary for formation of the TZ or for BB/TZ anchoring to the membrane during early ciliogenesis, but after these events IFT proteins participate in building (and maintaining) the rest of the ciliary axoneme, and BBS proteins participate in the delivery of ciliary cargo. A hierarchical, modular interaction network involving TZ proteins We propose that MKS/MKSR and NPHP proteins form a hierarchical network comprised of distinct modules with partial functional redundancy (Fig. 10, B and C). The rationale for this modular hypothesis stems from our findings that in C. elegans, synthetic ciliary defects result from mutations in at least two TZ genes—but not any two genes (Fig. 3, B–D; Figs. 4–7). For example, even triple B9 gene (mks-1, mksr-1, and mksr-2) or nphp-1;nphp-4 double mutants lack prominent (additive) ciliary defects (Jauregui et al., 2008; Bialas et al., 2009). In contrast, disrupting any B9 gene together with nphp-1 or nphp-4 results in severe cilia anomalies (Williams et al., 2008). Other mutant combinations such as mks-1;mks-3, or mks-6;mksr-2, do not noticeably disrupt cilia, whereas the same four individual mutants show strong genetic interactions with nphp-1 or nphp-4 (Fig. 3, B–D; Figs. 4–7). Thus, we group MKS-1, MKSR-1, MKSR-2, MKS-3, and MKS-6 into an MKS/MKSR module and NPHP-1 and NPHP-4 into an NPHP module (Fig. 10 C). Importantly, MKS-5 represents an exception to this module assignment. Disrupting mks-5 together with mksr-1, mksr-2, mks-6 (MKS/MKSR module), or nphp-4 (NPHP module) compromises cilia morphology (Fig. 3, B–D). Moreover, mks-5 mutants fail to localize MKS-1, MKSR-1, MKSR-2, MKS-3, and MKS-6 (i.e., the entire known MKS/MKSR module) to the TZ; the mks-5 mutation also partially alters NPHP-1 and NPHP-4 distribution at the TZ (summarized in Fig. 10 B). Based on our data, we propose that the MKS/MKSR and NPHP modules are functionally linked through MKS-5 (Fig. 10 C). Whether the modules are physically associated together at the TZ remains to be examined; however, the established biochemical interaction between mammalian MKS5/RPGRIP1L and NPHP4 (Roepman et al., 2005) indicates this is likely. Further, the likelihood that MKS-5 links multiple functional modules is supported by the finding that human RPGRIP1L mutations contribute to at least six distinct ciliopathies, including MKS and NPHP (Zaghloul and Katsanis, 2010). The genetic interactions we observe in C. elegans also likely exist between TZ genes in mammals. For example, RPGRIP1L and CEP290 heterozygous variants are linked to increased phenotypic pleiotropy in some NPHP patients in which primary disease symptoms are caused by mutations in other genes (Tory et al., 2007; Khanna et al., 2009). Our findings may also help explain data reported by Hoefele et al. (2005), who screened human NPHP patients and determined that single heterozygous mutations in NPHP4 were over three times more prevalent than two recessive mutations. Based on our genetic interaction studies in C. elegans, we predict that the ailments in these patients result from interactions between mutations in other TZ module genes. With the growing number of available MKS and NPHP rodent models, such possibilities could be formally examined by assessing the consequences of combining mutant alleles of multiple TZ genes. Intriguingly, proteins seemingly absent in C. elegans or Drosophila have recently been implicated in BB- or TZ-associated functions in other organisms. These include the Ahi1 ciliopathy protein, which binds RAB8 and is proposed to play a role in polarized membrane trafficking (Hsiao et al., 2009), as well as OFD1 and Talpid3, which may assist with docking the ciliary vesicle onto the centriole (Yin et al., 2009; Singla et al., 2010). Another such protein is CEP290, which binds MKS6 (Gorden et al., 2008) and thus likely functions in the MKS or NPHP module. CEP290 was assigned as a component of Y-links and ciliary gate in Chlamydomonas (Craige et al., 2010), and mutations in this gene occur in several ciliopathies. Whether CEP290 functionally interacts with other TZ proteins to facilitate BB/TZ membrane attachments in an early ciliogenic event—as we have shown in this study for several evolutionarily conserved TZ proteins—remains to be determined. TZ vs. BB The TZ is an underappreciated ciliary subcompartment, often incorrectly presumed to be one and the same with the adjacent BB. This misconception was inadvertently strengthened by Keller et al. (2005), who identified constituents of the Chlamydomonas “basal body” proteome; however, in these analyses the TZ was co-isolated with the BB. Thus, some TZ proteins (e.g., CEP290, NPHP4) were ascribed to the BB, leading to the widespread belief that NPHP and MKS are diseases of BB dysfunction. Confusion regarding differences between the C. elegans BB and TZ is also apparent. As C. elegans lacks classical BB microtubule architecture at the base of cilia (Perkins et al., 1986), similar to that observed in mature murine sperm (Manandhar et al., 1998), it has long been speculated that the nematode TZ is “analogous” to the BB. However, several observations suggest this is inaccurate. First, in daf-19 mutants, which lack TZs and cilia, typical centriolar pairs are observed unattached to distal dendritic membranes where cilia would normally form (Perkins et al., 1986). Second, TFs present at the base of C. elegans cilia (Fig. 1 A; Perkins et al., 1986) are evolutionarily conserved structures present at the distal end of all BBs (Silverman and Leroux, 2009). Third, the conserved centriolar/BB protein HYLS-1 localizes in C. elegans within the TF region, just proximal to NPHP-4 at the TZ (Dammermann et al., 2009). Importantly, HYLS-1 in C. elegans and vertebrates is dispensable for centriole function during cell division but critical for ciliogenesis (Dammermann et al., 2009). We propose that the C. elegans TF region, where different IFT proteins concentrate—as shown for IFT52 in Chlamydomonas (Deane et al., 2001)—functions as a bona fide BB that is adjacent to but distinct from the TZ, where MKS/MKSR/NPHP proteins localize (Fig. 1 A, B–D; Fig. 2). Further, our finding that Y-links are completely, or even selectively, lost in MKS/MKSR/NPHP double mutants leaves open the possibility that at least some of these proteins represent structural elements (e.g., Y-links) of the TZ. Indeed, structural and biochemical properties of various MKS/MKSR/NPHP proteins support this possibility; NPHP1 binds microtubules (Otto et al., 2003), the C2/B9 domain proteins are predicted to associate with the inner leaflet of the plasma membrane, and MKS3 is membrane spanning. Taken together, one begins to envision two or more MKS/MKSR/NPHP modules—likely joined by MKS5—as components of the Y-link/ciliary necklace structures that connect axonemal microtubules to the surrounding membrane. TZ-dependent establishment of the ciliary gate Our analyses revealed that MKS/MKSR and NPHP modules are collectively required for two essential aspects of ciliogenesis, namely membrane anchoring of the BB/TZ and formation of an intact TZ region. Through these processes, the ciliary gate is established (Fig. 10 D). The observed axoneme extension defects in TZ double mutants probably arise as a secondary consequence of anomalies in these early ciliogenic events. The TZ in early ciliogenesis. Largely normal IFT rates in TZ mutant strains, and the fact that IFT mutants do not display phenotypes comparable to those of TZ mutants by TEM analysis (Perkins et al., 1986), support the notion that IFT proteins play a role in ciliogenesis distinct from that of TZ proteins, namely building the axoneme and delivering ciliary cargo vs. microtubule-membrane association/stabilization (Fig. 10 D). This difference is reflected in rodent models in which IFT perturbation (cilia ablation) causes early embryonic lethality (at ∼E8.5–10.5), whereas disrupting MKS/MKSR/NPHP proteins leads to less severe ciliogenesis defects and developmental outcomes (Murcia et al., 2000; Jiang et al., 2009; Tammachote et al., 2009). TEM studies of mammalian cells forming cilia (Sorokin, 1962) are consistent with a possible role for the TZ in early ciliogenesis (Fig. 10 D). Specifically, an early ciliogenic step involves the interaction of a Golgi-derived “ciliary” vesicle (CV) with the distal end of the mother centriole/nascent BB to establish microtubule–membrane associations. A “ciliary bud” grows from the centriole, invaginating the CV, which itself appears to grow by fusion with secondary vesicles. In all likelihood, the ciliary bud, visible before BB docking/anchoring with the plasma membrane (Sorokin, 1962; Moser et al., 2010), represents a developing TZ; i.e., the first section of the ciliary axoneme (Fig. 10 D; Rohatgi and Snell, 2010). After docking and fusion with the membrane, the ciliary bud/TZ further extends to form the ciliary axoneme, a step dependent on IFT proteins. Alternatively, in other cell types, the centriole/TZ may dock directly with the plasma membrane, forgoing association with a CV; in this instance, microtubule–membrane associations mediated through the TZ also occur before axoneme extension (Fig. 10 D). The TZ ciliary gate. The ciliary gate was long thought to be important in cilia function (Rosenbaum and Witman, 2002), but is only now being studied at the molecular level (Jauregui et al., 2008; Craige et al., 2010). The BB/TZ region is thought to facilitate ciliary gate function as a docking site for proteins destined for the cilium, as a region of selective active transport, and as a diffusion barrier. We demonstrate that individual MKS/MKSR and NPHP-disrupted strains lack a normal ciliary barrier, as evidenced by accumulation of nonciliary proteins within cilia. Alternatively, these proteins may enter cilia at low levels normally and instead require NPHP and MKS proteins for efficient removal from the compartment. Regardless, our data indicate that MKS/MKSR/NPHP proteins establish the TZ as a ciliary gate, and we predict that these TZ proteins likely function in coordination with other mechanisms—including IFT, the Ran-importin system, and BBSome (Bae et al., 2006; Jauregui et al., 2008; Craige et al., 2010; Dishinger et al., 2010; Jin et al., 2010)—to control cilium composition and thus function. Concluding remarks Ciliopathies are genetically heterogeneous but have overlapping phenotypic presentations, suggesting a common cellular mechanism as the basis of their etiology. In this study, we show that eight C. elegans proteins jointly function in establishing connections between the ciliary membrane and axoneme at the TZ, and in formation of the ciliary gate to regulate ciliary membrane composition. These TZ proteins are highly conserved in ciliated organisms (Hodges et al., 2010), suggesting that our model will be widely applicable. Materials and methods Strains and general methods All strains (Table S1 B) used were maintained and cultured at 20°C using standard techniques (Brenner, 1974). Many procedures used in this study are summarized in Inglis et al. (2009). K07G5.3(gk674) and C09G5.8(tm3100) worms were obtained from the knockout consortium (http://celeganskoconsortium.omrf.org) and the National Bioresource Project (Japan), respectively, and outcrossed 5x to wild type. Because the gk674 deletion removes some neighboring xpa-1 gene sequence (Fig. 3 A), we confirmed that the nonciliary function of this DNA repair gene was not abrogated. First, the lifespan of the available xpa-1(mn157) mutant is shorter (P
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VDAC2 inhibits BAK activation and mitochondrial apoptosis.
Emily H.-Y. Cheng, Tatiana V Sheiko, Jill Fisher … (2003)
The multidomain proapoptotic molecules BAK or BAX are required to initiate the mitochondrial pathway of apoptosis. How cells maintain the potentially lethal proapoptotic effector BAK in a monomeric inactive conformation at mitochondria is unknown. In viable cells, we found BAK complexed with mitochondrial outer-membrane protein VDAC2, a VDAC isoform present in low abundance that interacts specifically with the inactive conformer of BAK. Cells deficient in VDAC2, but not cells lacking the more abundant VDAC1, exhibited enhanced BAK oligomerization and were more susceptible to apoptotic death. Conversely, overexpression of VDAC2 selectively prevented BAK activation and inhibited the mitochondrial apoptotic pathway. Death signals activate "BH3-only" molecules such as tBID, BIM, or BAD, which displace VDAC2 from BAK, enabling homo-oligomerization of BAK and apoptosis. Thus, VDAC2, an isoform restricted to mammals, regulates the activity of BAK and provides a connection between mitochondrial physiology and the core apoptotic pathway.
Author and article information
Contributors
Gang Dong: Role: Academic Editor
William Tsang: Role: Academic Editor
Journal
Journal ID (nlm-ta): Cells
Journal ID (iso-abbrev): Cells
Journal ID (publisher-id): cells
Title:
Cells
Publisher:
MDPI
ISSN
(Electronic):
2073-4409
Publication date
(Electronic):
31
July
2015
Publication date Collection:
September
2015
Volume: 4
Issue: 3
Pages: 331-353
Affiliations
Department of Molecular Genetics, The Ohio State University, 484 W. 12th Avenue, Columbus,
OH 43210-1292, USA; E-Mails:
majumder.7@
123456osu.edu
(S.M.);
aaecash@
123456gmail.com
(A.C.)
Author notes
[*
]Author to whom correspondence should be addressed; E-Mail:
fisk.13@
123456osu.edu
; Tel.: +1-614-292-0318; Fax: +1-614-292-4466.
Article
Publisher ID: cells-04-00331DOI: 10.3390/cells4030331
PMC ID: 4588040
PubMed ID: 26264029
SO-VID: 8284a45e-0597-41ce-b9b7-b264ab9d0376
Copyright © © 2015 by the authors; licensee MDPI, Basel, Switzerland.
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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).
History
Date
received
: 04
June
2015
Date
accepted
: 27
July
2015
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