Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

Erythropoietin Receptor (EpoR) Agonism Is Used to Treat a Wide Range of Disease

The erythropoietin receptor (EpoR) was discovered and described in red blood cells (RBCs), stimulating its proliferation and survival. The target in humans for EpoR agonists drugs appears clear—to treat anemia. However, there is evidence of the pleitropic actions of erythropoietin (Epo). For that reason, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson’s and Alzheimer’s disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia). Unfortunately, the side effects of rhEpo are also evident. A new generation of nonhematopoietic EpoR agonists drugs (asialoEpo, Cepo and ARA 290) have been investigated and further developed. These EpoR agonists, without the erythropoietic activity of Epo, while preserving its tissue-protective properties, will provide better outcomes in ongoing clinical trials. Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.In principle, the erythropoietin receptor (EpoR) was discovered and described in red blood cell (RBC) progenitors, stimulating its proliferation and survival. Erythropoietin (Epo) is mainly synthesized in fetal liver and adult kidneys (1–3). Therefore, it was hypothesized that Epo act exclusively on erythroid progenitor cells. Accordingly, the target in humans for EpoR agonists drugs (such as recombinant erythropoietin [rhEpo], in general, called erythropoiesis-simulating agents) appears clear (that is, to treat anemia). However, evidence of a kaleidoscope of pleitropic actions of Epo has been provided (4,5). The Epo/EpoR axis research involved an initial journey from laboratory basic research to clinical therapeutics. However, as a consequence of clinical observations, basic research on Epo/EpoR comes back to expand its clinical therapeutic applicability.Although kidney and liver have long been considered the major sources of synthesis, Epo mRNA expression has also been detected in the brain (neurons and glial cells), lung, heart, bone marrow, spleen, hair follicles, reproductive tract and osteoblasts (6–17). Accordingly, EpoR was detected in other cells, such as neurons, astrocytes, microglia, immune cells, cancer cell lines, endothelial cells, bone marrow stromal cells and cells of heart, reproductive system, gastrointestinal tract, kidney, pancreas and skeletal muscle (18–27). Conversely, Sinclair et al.(28) reported data questioning the presence or function of EpoR on nonhematopoietic cells (endothelial, neuronal and cardiac cells), suggesting that further studies are needed to confirm the diversity of EpoR. Elliott et al.(29) also showed that EpoR is virtually undetectable in human renal cells and other tissues with no detectable EpoR on cell surfaces. These results have raised doubts about the preclinical basis for studies exploring pleiotropic actions of rhEpo (30).For the above-mentioned data, a return to basic research studies has become necessary, and many studies in animal models have been initiated or have already been performed. The effect of rhEpo administration on angiogenesis, myogenesis, shift in muscle fiber types and oxidative enzyme activities in skeletal muscle (4,31), cardiac muscle mitochondrial biogenesis (32), cognitive effects (31), antiapoptotic and antiinflammatory actions (33–37) and plasma glucose concentrations (38) has been extensively studied. Neuro- and cardioprotection properties have been mainly described. Accordingly, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson’s and Alzheimer’s disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia).Unfortunately, the side effects of rhEpo are also evident. Epo is involved in regulating tumor angiogenesis (39) and probably in the survival and growth of tumor cells (25,40,41). rhEpo administration also induces serious side effects such as hypertension, polycythemia, myocardial infarction, stroke and seizures, platelet activation and increased thromboembolic risk, and immunogenicity (42–46), with the most common being hypertension (47,48). A new generation of nonhematopoietic EpoR agonists drugs have hence been investigated and further developed in animals models. These compounds, namely asialoerythropoietin (asialoEpo) and carbamylated Epo (Cepo), were developed for preserving tissue-protective properties but reducing the erythropoietic activity of native Epo (49,50). These drugs will provide better outcome in ongoing clinical trials. The advantage of using nonhematopoietic Epo analogs is to avoid the stimulation of hematopoiesis and thereby the prevention of an increased hematocrit with a subsequent procoagulant status or increased blood pressure. In this regard, a new study by van Rijt et al. has shed new light on this topic (51). A new nonhematopoietic EpoR agonist analog named ARA 290 has been developed, promising cytoprotective capacities to prevent renal ischemia/reperfusion injury (51). ARA 290 is a short peptide that has shown no safety concerns in preclinical and human studies. In addition, ARA 290 has proven efficacious in cardiac disorders (52,53), neuropathic pain (54) and sarcoidosis-induced chronic neuropathic pain (55). Thus, ARA 290 is a novel nonhematopoietic EpoR agonist with promising therapeutic options in treating a wide range of pathologies and without increased risks of cardiovascular events.Overall, this new generation of EpoR agonists without the erythropoietic activity of Epo while preserving tissue-protective properties of Epo will provide better outcomes in ongoing clinical trials (49,50). Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases, such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.     

Dynein at the nuclear envelope

Most cellular organelles are positioned through active transport by motor proteins. The authors discuss the evidence that dynein has important cell cycle-regulated functions in this context at the nuclear envelope.Most cellular organelles are positioned through active transport by motor proteins. This is especially important during cell division, a time when the organelles and genetic content need to be divided equally between the two daughter cells. Although individual proteins can attain their correct location by diffusion, larger structures are usually positioned through active transport by motor proteins. The main motor that transports cargoes to the minus ends of the microtubules is dynein. In nondividing cells, dynein probably transports or positions the nucleus inside the cells by binding to the nuclear envelope (NE; Burke & Roux, 2009). However, it appears that dynein also has important cell-cycle-regulated functions at the NE, as it is recruited to the NE every cell cycle just before cells enter mitosis (Salina et al, 2002; Splinter et al, 2010). Here, we discuss why dynein might be recruited to the NE for a brief period before mitosis.During late G2 or prophase the centrosomes separate to opposite sides of the nucleus, but remain closely associated with the NE during separation. This close association is probably mediated through NE-bound dynein, which ‘walks'' towards the minus ends of centrosomal microtubules, thereby pulling centrosomes towards the NE (Splinter et al, 2010; Gonczy et al, 1999; Robinson et al, 1999). We speculate that close association of centrosomes to the NE might have several functions. First, if centrosomes are not mechanically coupled to the NE, centrosome movement during separation will occur in random directions and chromosomes will not end up between the two separated centrosomes. In this scenario, individual kinetochores might attach more frequently to microtubules coming from both centrosomes (merotelic attachments), a defect that can result in aneuploidy, a characteristic of cancer. Second, centrosome-nuclear attachment also keeps centrosomes in close proximity to chromosomes, which might facilitate rapid capture of chromosomes by microtubules nucleated by the centrosomes after NE breakdown. This might not be absolutely essential, as chromosome alignment can occur in the absence of centrosomes. However, the spatial proximity of centrosomes and chromosomes at NE breakdown might improve the fidelity of kinetochore capture and chromosome alignment.In addition, dynein has also been suggested to promote centrosome separation in prophase in some systems (Gonczy et al, 1999; Robinson et al, 1999; Vaisberg et al, 1993), although not in others (Tanenbaum et al, 2008). Perhaps dynein, anchored at the NE just before mitosis, could exert force on microtubules emanating from both centrosomes, thereby pulling centrosomes apart. However, this force could also be produced by cortical dynein and specific inhibition of NE-associated or cortical dynein will be required to test which pool is responsible.Dynein has also been implicated in the process of NE breakdown itself, by promoting mechanical shearing of the NE. Two elegant studies showed that microtubules can tear the NE as cells enter mitosis (Salina et al, 2002; Beaudouin et al, 2002). One possibility is that microtubules growing into the NE mechanically disrupt it. Alternatively, NE-associated dynein might ‘walk'' along centrosomal microtubules and thereby pull on the NE, tearing it apart. However, testing the exact role of dynein in NE breakdown is complicated by the fact that centrosomes detach from the NE on inactivation of dynein and centrosomal microtubules stop growing efficiently into the NE. Thus, selective inhibition of dynein function will also be required to test this idea.Specific recruitment of dynein to the NE just before mitosis clearly suggests a role for dynein at the NE in preparing cells for mitosis. A major role of NE-associated dynein is to maintain close association of centrosomes with the NE during centrosome separation, which might be needed for efficient capture and alignment of chromosomes after NE breakdown, but additionally, NE-associated dynein could facilitate breakdown and contribute to centrosome separation in some systems.     

Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase

EMBO J 32 15, 2099–2112 doi:10.1038/emboj.2013.125; published online May312013Mutations in Parkin represent ∼50% of disease-causing defects in autosomal recessive-juvenile onset Parkinson''s disease (AR-JP). Recently, there have been four structural reports of autoinhibited forms of this RING-IBR-RING (RBR) ubiquitin ligase (E3) by the Gehring, Komander, Johnston and Shaw groups. The important advances from these studies set the stage for the next steps in understanding the molecular basis for Parkinson''s disease (PD).Regulated protein degradation requires that E3s and their access to substrates be exquisitely controlled. RBR family E3s provide striking examples of this regulation. The complex and compact structures of Parkin (Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) as well as another RBR E3, human homologue of Ariadne (HHARI) (Duda et al, 2013), demonstrate extraordinarily intricate inter-domain arrangements. These autoinhibited structures ensure that their functions are restricted until activated.Until recently, RBR E3s were believed to be a subclass of RING E3s, which allosterically activate E2 conjugated with ubiquitin (E2∼Ub). However, Wenzel et al (2011) determined that they are actually hybrid E3s, containing an E2 binding site in RING1 and a catalytic cysteine residue in the domain designated as RING2. The catalytic cysteine is an acceptor for an ubiquitin from RING1-bound E2∼Ub forming an intermediate (E3∼Ub) that leads to substrate or autoubiquitination. In this way, RBRs resemble HECT E3s, which also form catalytic intermediates in ubiquitination. There are 13 human RBR family E3s. Besides Parkin, two notable RBRs are HOIL-1 and HOIP, which form part of a complex integral to NF-κB activation (Wenzel and Klevit, 2012).In addition to causal roles in AR-JP, single allele mutations of Parkin are found in some sporadic cases of PD (references in Wauer and Komander, 2013). Mutations in the Parkin-associated kinase PINK1, which is upstream of Parkin, also account for a significant number of AR-JP cases (Hardy et al, 2009; Narendra et al, 2012; Lazarou et al, 2013). A number of diverse Parkin substrates have been postulated to be associated with PD. There is substantial evidence that one role for Parkin is at mitochondria. Once activated and recruited to damaged/depolarized mitochondria by PINK1, it ubiquitinates exposed mitochondrial proteins leading to both proteasomal degradation and mitophagy (Narendra et al, 2012; Sarraf et al, 2013). Parkin has also been implicated in cell surface signalling and as a tumour suppressor (see references in Wauer and Komander, 2013).Parkin encodes five structured domains, beginning with an N-terminal ubiquitin-like domain (UbLD) and followed by four domains that each bind two zinc (Zn) atoms (Figure 1A). The most N-terminal of the Zn-binding domains is RING0. C-terminal to this is the RBR, consisting of RING1, the IBR and RING2. The crystal structures of inactive Parkin from Riley et al (2013), Trempe et al (2013) and Wauer and Komander (2013) show remarkable congruity. Spatially, the IBR is at the complete opposite end of the molecule from RING2, to which it is connected by a partially unstructured ∼37 residue linker. This linker includes a two-turn helix, referred to as the repressor element of Parkin (REP) or tether, which binds and occludes the E2 binding face of RING1. RING1 occupies the central position in these structures, and RING0 separates RING1 from RING2 (Figure 1B and C). The latter contains the residue identified by Wenzel et al (2011), and confirmed by all three groups, to be the catalytic cysteine, C431. A lower resolution structure also includes the UbLD and places this domain adjacent to RING1 (Trempe et al, 2013). A second unstructured linker connects the UbLD and RING0. UbLDs are involved in a number of protein–protein interactions and small angle X-ray scattering confirms that this domain is integral to the core structure of Parkin (Spratt et al, 2013; Trempe et al, 2013). Biophysical characterization of Parkin and HHARI suggests that each is a monomer in solution.Open in a separate windowFigure 1Schematic and spatial representation of Parkin. (A) Primary structure and domain designations of Parkin, including the REP sequence within the otherwise unstructured IBR-RING2 linker. (B) Structural representation of full-length Parkin (PDB 4K95) highlighting the complex domain interactions in the three-dimensional structure, the catalytic C431 residue, and residue W403 within the REP, which plays a role in stabilizing the autoinhibited form of Parkin. (C) A model of Parkin with the E2 UbcH5B/Ube2D2 bound (devised using PDB 4K95 and PDB 4AP4 to mimic the position of an E2 bound to RING1) to illustrate the required displacement of UbLD and REP and the large distance between the E2∼Ub attachment site of the E2 and the catalytic active site of Parkin. Note that in this conformation the catalytic Cys within RING2 (C431) remains buried by RING0.RING1 is the only bona fide RING domain. All NMR and crystal structures of IBR domains from Parkin, HHARI and HOIP (PDB ID: 2CT7) are in good agreement. The Parkin and HHARI RING2s are structurally highly homologous and share a common Zn-coordinating arrangement with IBR domains. In contrast to the IBR and RING2, RING0 has a distinct arrangement of Zn-coordinating residues (Beasley et al, 2007; Duda et al, 2013; Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) (see Figure 1F of Trempe et al (2013) for the various Zn coordination arrangements).All of the Parkin crystal structures represent inactive forms of the E3. This is imposed by the quaternary positioning of the domains, which precludes activity in multiple ways. RING0 plays two obvious roles to maintain Parkin in an inactive state. RING0 shares an interface with RING2 and buries C431, making it unavailable as an ubiquitin acceptor. Moreover, RING0 intervenes between RING1 and RING2, creating an insurmountable separation of >50 Å between the active site Cys of an E2 bound to RING1 and C431 (Figure 1B and C). Thus, RING0 must be displaced for ubiquitin transfer to occur. Accordingly, deletion of RING0 results in a marked increase in Parkin autoubiquitination and in C431 reactivity (Riley et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013). In HHARI, these two inhibitory functions are fulfilled by the C-terminal Ariadne domain, which similarly interposes between RING1 and RING2 (Duda et al, 2013).Additional inhibition is provided by the REP, which binds to RING1 at the canonical RING-E2 binding site and prevents E2 binding. This provides at least a partial explanation for the impaired ability of Parkin to bind E2 when compared to HHARI, which lacks this element (Duda et al, 2013). A disease-associated REP mutant (A398T) at the RING1 interface increases autoubiquitination (Wauer and Komander, 2013). The significance of inhibition by REP-RING1 binding was verified by mutating a critical RING1-interacting REP residue (W403A). This increased autoubiquitination and E2 binding (Trempe et al, 2013). Consistent with the requirement for charging C431 with ubiquitin in mitochondrial translocation (Lazarou et al, 2013), Parkin association with depolarized mitochondria is accelerated with this mutation (Trempe et al, 2013). Interestingly, W403 also interacts with the C-terminal Val of Parkin within RING2, and could therefore potentially further stabilize the autoinhibited form of the protein (Riley et al, 2013), consistent with previous observations (Henn et al, 2005).The quaternary structure of full-length Parkin also suggests that displacement of its N-terminal UbLD must occur for full activation (Trempe et al, 2013). The positioning of the UbLD adjacent to RING1 indicates that it would provide a steric impediment to E2∼Ub binding (Figure 1B and C). Additionally, displacement of the UbLD could be important to relieve interactions with the IBR-RING2 linker, which, as suggested in a previous study (Chaugule et al, 2011), might help to maintain Parkin in an inactive state. Finally, the crystal structure of the full-length Parkin indicates that the UbLD is not available for interactions with other proteins. This would limit Parkin''s range of intermolecular interactions.RBR E3s have at least two domains critical for sequential ubiquitin transfer and full activity, RING1 and RING2. The RING1 of Parkin, as well as all other RBR E3s, is notable in lacking the basic residue in the second Zn coordinating loop (or its equivalent in U-box proteins), which has recently been implicated in RING-mediated transfer of Ub from E2∼Ub (Metzger et al, 2013). This suggests that other factors play compensatory roles in positioning ubiquitin for transfer from E2∼Ub to C431. A non-mutually exclusive possibility is that the lack of this basic residue in RING1 limits unwanted attack on the E2∼Ub linkage, thereby minimizing the unregulated ubiquitination. Turning to RING2, the area surrounding the active site C431 of Parkin is notable in that it includes a sequence recognizable as a catalytic triad, similar to that in deubiquitinating enzymes. The Cys-His-Glu grouping, found in Parkin and other RBR E3s, contributes to in vitro activity (Riley et al, 2013; Wauer and Komander, 2013). Interestingly, however, the Glu was dispensable in a cellular assay (Riley et al, 2013). This triad is conserved in HHARI, where an Asn between the Cys and His residues (found in a number of RBRs but not conserved in Parkin), was found to be important for catalysis (Duda et al, 2013).The advances made in these studies impart significant information about an important and clinically relevant E3. However, Parkin, as well as HHARI, has been captured in their inactive, unmodified forms. One obvious question is how does Parkin transition between inactive and active states. PINK1 is implicated in phosphorylating Parkin on its UbLD and potentially other sites, with evidence that phosphorylation contributes to Parkin activation (Narendra et al, 2012). How phosphorylation could contribute to protein interactions that might facilitate Parkin activation, potentially including Parkin oligomerization (Lazarou et al, 2013), is unknown. Regardless, it is evident that considerable unwinding of its quaternary structure must take place.While there is much work ahead to understand these processes, one important interface that must be disrupted for activation is that between the REP and RING1. It is intriguing to consider that such interruption might be associated with other alterations in the IBR-RING2 linker, potentially facilitating the movement of the UbLD from RING1 and contributing to activation. Related to activation is the all-important question of how Parkin recognizes and targets specific substrates. While the UbLD represents a potential site of interaction, most purported substrates are not known to have UbLD-interaction domains. Although interactions involving the UbLD could occur indirectly, through bridging molecules, there is also evidence that other regions of Parkin, including the RBR region, might recognize substrates either directly or indirectly (Tsai et al, 2003) and that some substrates may be phosphorylated by PINK1 (Narendra et al, 2012). Conformational changes induced by substrate interactions, particularly in the IBR RING2 linker, could, as above, represent an important aspect of activation.There are over 75 missense mutations of Parkin associated with AR-JP, most of these inactivate the protein, but there are also some that are activating (Wauer and Komander, 2013). Activating mutations presumably result in pathology at least partially as a consequence of increased autoubiquitination and degradation (e.g., A398T). The current studies help to provide a classification of missense mutations into those that affect (i) folding or stability, (ii) catalytic mechanism, and (iii) interactions between domains. Interdomain mutations might inactivate or contribute to constitutive activation leading to autoubiquitination and degradation.Finally, we know little about how the autosomal recessive and the much more prevalent sporadic forms of PD overlap in their molecular pathology. However, mitochondrial dysfunction is increasingly a common theme. Thus, with the structure of the inactive protein in hand, there is hope that we can begin to consider ways in which domain interactions might be altered in a controlled manner to activate, but not hyperactivate, this critical E3 and lessen the progression of PD.     

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).The textbook notion that apoptosis would always take place unrecognized by the immune system has recently been invalidated (Zitvogel et al, 2010; Galluzzi et al, 2012). Thus, in specific circumstances (in particular in response to anthracyclines, oxaliplatin, and γ irradiation), cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumour-specific immune responses (Zitvogel et al, 2010). These signals include the pre-apoptotic exposure of intracellular proteins such as the endoplasmic reticulum (ER) chaperon CRT and the heat-shock protein HSP90 at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1 (Zitvogel et al, 2010). Together, these processes (and perhaps others) constitute the molecular determinants of ICD.In this issue of The EMBO Journal, Garg et al (2012) add hypericin-based PTD (Hyp-PTD) to the list of bona fide ICD inducers and convincingly link Hyp-PTD-elicited ICD to the functional activation of the immune system. Moreover, Garg et al (2012) demonstrate that Hyp-PDT stimulates ICD via signalling pathways that overlap with—but are not identical to—those elicited by anthracyclines, which constitute the first ICD inducers to be characterized (Casares et al, 2005; Zappasodi et al, 2010; Fucikova et al, 2011).Intrigued by the fact that the ER stress response is required for anthracycline-induced ICD (Panaretakis et al, 2009), Garg et al (2012) decided to investigate the immunogenicity of Hyp-PDT (which selectively targets the ER). Hyp-PDT potently stimulated CRT exposure and ATP release in human bladder carcinoma T24 cells. As a result, T24 cells exposed to Hyp-PDT (but not untreated cells) were engulfed by Mf4/4 macrophages and human dendritic cells (DCs), the most important antigen-presenting cells in antitumour immunity. Similarly, murine colon carcinoma CT26 cells succumbing to Hyp-PDT (but not cells dying in response to the unspecific ER stressor tunicamycin) were preferentially phagocytosed by murine JAWSII DCs, and efficiently immunized syngenic BALB/c mice against a subsequent challenge with living cells of the same type. Of note, contrarily to T24 cells treated with lipopolysaccharide (LPS) or dying from accidental necrosis, T24 cells exposed to Hyp-PDT activated DCs while eliciting a peculiar functional profile, featuring high levels of NO production and absent secretion of immunosuppressive interleukin-10 (IL-10) (Garg et al, 2012). Moreover upon co-culture with Hyp-PDT-treated T24 cells, human DCs were found to secrete high levels of IL-1β, a cytokine that is required for the adequate polarization of interferon γ (IFNγ)-producing antineoplastic CD8⁺ T cells (Aymeric et al, 2010). Taken together, these data demonstrate that Hyp-PDT induces bona fide ICD, eliciting an antitumour immune response.By combining pharmacological and genetic approaches, Garg et al (2012) then investigated the molecular cascades that are required for Hyp-PDT-induced CRT exposure and ATP release. They found that CRT exposure triggered by Hyp-PDT requires reactive oxygen species (as demonstrated with the ¹O₂ quencher L-histidine), class I phosphoinositide-3-kinase (PI3K) activity (as shown with the chemical inhibitor wortmannin and the RNAi-mediated depletion of the catalytic PI3K subunit p110), the actin cytoskeleton (as proven with the actin inhibitor latrunculin B), the ER-to-Golgi anterograde transport (as shown using brefeldin A), the ER stress-associated kinase PERK, the pro-apoptotic molecules BAX and BAK as well as the CRT cell surface receptor CD91 (as demonstrated by their knockout or RNAi-mediated depletion). However, there were differences in the signalling pathways leading to CRT exposure in response to anthracyclines (Panaretakis et al, 2009) and Hyp-PDT (Garg et al, 2012). In contrast to the former, the latter was not accompanied by the exposure of the ER chaperon ERp57, and did not require eIF2α phosphorylation (as shown with non-phosphorylatable eIF2α mutants), caspase-8 activity (as shown with the pan-caspase blocker Z-VAD.fmk, upon overexpression of the viral caspase inhibitor CrmA and following the RNAi-mediated depletion of caspase-8), and increased cytosolic Ca²⁺ concentrations (as proven with cytosolic Ca²⁺ chelators and overexpression of the ER Ca²⁺ pump SERCA). Moreover, Hyp-PDT induced the translocation of CRT at the cell surface irrespective of retrograde transport (as demonstrated with the microtubular poison nocodazole) and lipid rafts (as demonstrated with the cholesterol-depleting agent methyl-β-cyclodextrine). Of note, ATP secretion in response to Hyp-PDT depended on the ER-to-Golgi anterograde transport, PI3K and PERK activity (presumably due to their role in the regulation of secretory pathways), but did not require BAX and BAK (Garg et al, 2012). Since PERK can stimulate autophagy in the context of ER stress (Kroemer et al, 2010), it is tempting to speculate that autophagy is involved in Hyp-PDT-elicited ATP secretion, as this appears to be to the case during anthracycline-induced ICD (Michaud et al, 2011).Altogether, the intriguing report by Garg et al (2012) demonstrates that the stress signalling pathways leading to ICD depend—at least in part—on the initiating stimulus (Figure 1). Speculatively, this points to the coexistence of a ‘core'' ICD signalling pathway (which would be common to several, if not all, ICD inducers) with ‘private'' molecular cascades (which would be activated in a stimulus-dependent fashion). Irrespective of these details, the work by Garg et al (2012) further underscores the importance of anticancer immune responses elicited by established and experimental therapies.Open in a separate windowFigure 1Molecular mechanisms of immunogenic cell death (ICD). At least three processes underlie the immunogenicity of cell death: the pre-apoptotic exposure of calreticulin (CRT) at the cell surface, the secretion of ATP, and the post-apoptotic release of HMGB1. ICD can be triggered by multiple stimuli, including photodynamic therapy, anthracycline-based chemotherapy, and some types of radiotherapy. The signalling pathways elicited by distinct ICD inducers overlap, but are not identical. In red are indicated molecules and processes that—according to current knowledge—may be required for CRT exposure and ATP secretion in response to most, if not all, ICD inducers. The molecular determinants of the immunogenic release of HMGB1 remain poorly understood. ER, endoplasmic reticulum; P-eIF2α, phosphorylated eIF2α; PI3K, class I phosphoinositide-3-kinase; ROS, reactive oxygen species.     

Cilia born out of shock and stress

EMBO J (2013) 32 23, 3029–3040 10.1038/emboj.2013.223; published online October112013Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins.Centrioles are barrel-shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule-organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle-dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule-dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM-1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet-Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: First, in cycling cells they may serve to sequester essential ciliary proteins (Stowe et al, 2012). Second, upon initiation of the ciliogenesis programme, centriolar satellite components seem to promote the recruitment of specific ciliary proteins to basal bodies (Ferrante et al, 2006; Lopes et al, 2011; Stowe et al, 2012).In a new study in The EMBO Journal, Villumsen et al (2013) now describe how stress–response pathways conspire to control ciliogenesis. The authors observed that specific environmental stresses, such as ultraviolet light radiation (UV) or heat shock, but not ionizing radiation (IR), trigger rapid displacement of PCM-1, AZI1 and CEP290 from centriolar satellites. However, OFD1 remained associated with satellites, indicating that centriolar satellites persist despite UV-induced removal of PCM-1. This might come as some surprise, since PCM-1 depletion by RNA interference (RNAi) is thought to disrupt satellite integrity (Kim et al, 2008; Lopes et al, 2011); however, satellite loss upon PCM-1 RNAi may be a consequence of prolonged depletion of PCM-1, while acute PCM-1 displacement by stress might only ‘remodel'' centriolar satellites. It is also possible that not all satellites are created equal, and they do vary in protein composition (Kim et al, 2008; Staples et al, 2012). If so, UV-induced PCM-1 removal may disrupt some, but not all satellites.A good candidate regulator of centriolar satellite remodelling was the stress-activated MAP kinase p38, and indeed, Villumsen et al (2013) found p38 MAPK activity to be stimulated by both UV and heat shock but not IR in U2OS cells, mirroring those very stress pathways that also cause displacement of AZI1 and PCM-1 from satellites. Furthermore, p38 MAPK was essential for UV-induced dispersal of PCM-1 and AZI1. The authors then tested the hypothesis that stress-induced centriolar satellite remodelling could involve changes in the interactome of AZI1, and—consistent with an earlier proteomics study (Akimov et al, 2011)—identified PCM-1, CEP290 and the mindbomb E3 ubiquitin protein ligase 1 (MIB1) as the main AZI1 binding partners. GFP-MIB1 localized to centriolar satellites and mono-ubiquitylated AZI1, PCM-1 and CEP290 in cycling cells. In response to UV, both ubiquitylation of these proteins and MIB1 activity were reduced; notably, UV-induced MIB1 inactivation was independent of p38 MAPK activity, indicating that these two enzymes may act via distinct pathways (Figure 1A).Open in a separate windowFigure 1(A) Regulation of centriolar satellite remodelling. (B) Schematic summary of how centriolar satellite remodelling might facilitate ciliogenesis. See text for details.What could be the purpose of MIB1-dependent ubiquitylation of these satellite proteins? It certainly does not seem to regulate subcellular targeting, as in MIB1-depleted cells, AZI1 and PCM-1 both localised normally to centriolar satellites and could still be displaced by UV. Instead, ubiquitylation seems to suppress the interaction between AZI1 and PCM-1, consistent with the observation that UV, a condition that also reduces their ubiquitylation, enhances the binding of AZI1 to PCM-1.PCM-1, CEP290 and AZI1 all participate in ciliogenesis (Kim et al, 2008; Wilkinson et al, 2009; Stowe et al, 2012), raising the possibility that MIB1 might also affect this process. Indeed, serum starvation, which is known to promote cilia formation, attenuated MIB1 activity. Furthermore, MIB1 overexpression reduced the ciliogenesis observed in serum-starved cells, while MIB1 depletion in proliferating cells triggered a marked increase in the proportion of cells that formed cilia; this seems to reflect a direct effect of MIB1 on ciliogenesis, since neither MIB1 depletion nor overexpression altered cell cycle progression. Taken together, downregulation of MIB1 enzymatic activity appears to be a pre-requisite for efficient ciliogenesis, regardless of whether it is triggered by physiological ciliogenesis-promoting signals or by environmental stresses, making MIB1 a novel negative regulator of cilia formation.The recent discovery of ciliopathy-associated mutations in constituents of the DNA damage response signalling pathway pointed to a connection between DNA damage and ciliogenesis (Chaki et al, 2012). With the new link between UV and centriolar satellites, the authors next asked if UV radiation might affect ciliogenesis. Remarkably, UV and heat shock both triggered cilia assembly in RPE-1 cells in a p38 MAPK-dependent manner. MIB1 depletion further enhanced ciliogenesis after UV radiation, again implying an additive effect of p38 MAPK signalling and MIB1 suppression (Figure 1A).While finer details on the precise role of centriolar satellite components in cilia formation are still lacking, a more coherent picture is finally starting to emerge. In cycling cells, ubiquitination by MIB1 could serve to limit the interaction between AZI1 and PCM-1 on centriolar satellites (Figure 1B). Under these conditions PCM-1 may bind and sequester CEP290, an essential ciliogenic protein, thereby precluding untimely cilia formation (Stowe et al, 2012). Both during normal and stress-induced ciliogenesis programs, remodelling of centriolar satellites creates a permissive environment for cilia formation, and a key step in this process is downregulation of MIB1 activity. While it remains to be established how the latter is achieved, it is clear that MIB1 inactivation causes loss of ubiquitylation and increased binding between AZI1 and PCM-1. Preferential interaction of PCM-1 with AZI1 could in turn facilitate release of CEP290 from centriolar satellites and its subsequent accumulation at the centrosome. Once CEP290 reaches the optimum concentration at the centriole/basal body, it could serve to tether AZI1–PCM-1 complexes. PCM-1 could then concentrate Rab8 GTPase near centrosomes, allowing CEP290 to recruit Rab8 into the cilium, where it acts to extend the ciliary membrane (Kim et al, 2008).Collectively, the findings reported here provide strong experimental support to the notion that centriolar satellites are negative regulators of ciliogenesis in proliferating cells. Their role is central to limit untimely formation of cilia in cells. Environmental strains elicit stress–response pathways that converge to relieve the ciliogenesis block imposed by satellites. It is tempting to speculate that stress-induced cilia might serve as signalling platforms and contribute to checkpoint activation or perhaps initiation of repair mechanisms, but more work is needed to establish the true purpose of ciliogenesis in this context. It is of considerable interest that a recent study reports that autophagy, another stress-induced pathway, selectively removes OFD1 from satellites to promote ciliogenesis (Tang et al, 2013). Therefore stress-mediated centriolar satellite remodelling seems to be an evolving theme in the control of ciliogenesis.     

The Amino-terminal Domain of Desmoplakin Binds to Plakoglobin and Clusters Desmosomal Cadherin–Plakoglobin Complexes

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.Desmosomes are highly organized adhesive intercellular junctions that couple intermediate filaments to the cell surface at sites of cell–cell adhesion (Farquhar and Palade, 1963; Staehelin, 1974; Schwarz et al., 1990; Garrod, 1993; Collins and Garrod, 1994; Cowin and Burke, 1996; Kowalczyk and Green, 1996). Desmosomes are prominent in tissues that experience mechanical stress, such as heart and epidermis, and the disruption of desmosomes or the intermediate filament system in these organs has devastating effects on tissue integrity (Steinert and Bale, 1993; Coulombe and Fuchs, 1994; Fuchs, 1994; McLean and Lane, 1995; Stanley, 1995; Bierkamp et al., 1996; Ruiz et al., 1996). Desmosomes are highly insoluble structures that can withstand harsh denaturing conditions (Skerrow and Matoltsy, 1974; Gorbsky and Steinberg, 1981; Jones et al., 1988; Schwarz et al., 1990). This property of desmosomes facilitated early identification of desmosomal components but has impaired subsequent biochemical analysis of the protein complexes that form between desmosomal components. Ultrastructurally, desmosomes contain a core region that includes the plasma membranes of adjacent cells and a cytoplasmic plaque that anchors intermediate filaments to the plasma membrane. The plaque can be further divided into an outer dense plaque subjacent to the plasma membrane and an inner dense plaque through which intermediate filaments appear to loop.Molecular genetic analysis has revealed that the desmosomal glycoproteins, the desmogleins and desmocollins, are members of the cadherin family of cell–cell adhesion molecules (for review see Buxton et al., 1993, 1994; Cowin and Mechanic, 1994; Kowalczyk et al., 1996). The classical cadherins, such as E-cadherin, mediate calcium-dependent, homophilic cell–cell adhesion (Nagafuchi et al., 1987). The mechanism by which the desmosomal cadherins mediate cell–cell adhesion remains elusive (Amagai et al., 1994; Chidgey et al., 1996; Kowalczyk et al., 1996), although heterophilic interactions have recently been detected between desmogleins and desmocollins (Chitaev and Troyanovsky, 1997). Both classes of the desmosomal cadherins associate with the cytoplasmic plaque protein plakoglobin (Kowalczyk et al., 1994; Mathur et al., 1994; Roh and Stanley, 1995b ; Troyanovsky et al., 1994), which is part of a growing family of proteins that share a repeated motif first identified in the Drosophila protein Armadillo (Peifer and Wieschaus, 1990). This multigene family also includes the desmosomal proteins band 6/plakophilin 1, plakophilin 2a and 2b, and p0071, which are now considered to comprise a subclass of the armadillo family of proteins (Hatzfeld et al., 1994; Heid et al., 1994; Schmidt et al., 1994; Hatzfeld and Nachtsheim, 1996; Mertens et al., 1996).The most abundant desmosomal plaque protein is desmoplakin, which is predicted to be a homodimer containing two globular end domains joined by a central α-helical coiled-coil rod domain (O''Keefe et al., 1989; Green et al., 1990; Virata et al., 1992). Previous studies have demonstrated that the carboxyl-terminal domain of desmoplakin interacts with intermediate filaments (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; Kouklis et al., 1994; Meng et al., 1997), and the amino-terminal domain of desmoplakin is required for desmoplakin localization to the desmosomal plaque (Stappenbeck et al., 1993). Direct evidence supporting a role for desmoplakin in intermediate filament attachment to desmosomes was provided recently when expression of an amino-terminal polypeptide of desmoplakin was found to displace endogenous desmoplakin from cell borders and disrupt intermediate filament attachment to the cell surface in A431 epithelial cell lines (Bornslaeger et al., 1996).The classical cadherins, such as E-cadherin, bind directly to both β-catenin and plakoglobin (Aberle et al., 1994; Jou et al., 1995; for review see Cowin and Burke, 1996). β-Catenin is also an armadillo family member (McCrea et al., 1991; Peifer et al., 1992), and both plakoglobin and β-catenin bind directly to α-catenin (Aberle et al., 1994, 1996; Jou et al., 1995; Sacco et al., 1995; Obama and Ozawa, 1997). α-Catenin is a vinculin homologue (Nagafuchi et al., 1991) and associates with both α-actinin and actin (Knudson et al., 1995; Rimm et al., 1995; Nieset et al., 1997). Through interactions with β- and α-catenin, E-cadherin is coupled indirectly to the actin cytoskeleton, and this linkage is required for the adhesive activity of E-cadherin (Ozawa et al., 1990; Shimoyama et al., 1992). In addition, E-cadherin association with plakoglobin appears to be required for assembly of desmosomes (Lewis et al., 1997), underscoring the importance of E-cadherin in the overall program of intercellular junction assembly. However, the hierarchy of molecular interactions that couple the desmosomal cadherins to the intermediate filament cytoskeleton is largely unknown, although the desmocollin cytoplasmic domain appears to play an important role in recruiting components of the desmosomal plaque (Troyanovsky et al., 1993, 1994). Since desmosomal cadherins form complexes with plakoglobin and because the amino-terminal domain of desmoplakin is required for desmoplakin localization at desmosomes, we hypothesized that the amino-terminal domain of desmoplakin interacts with the desmosomal cadherin– plakoglobin complex.In previous studies, we used L-cell fibroblasts to characterize plakoglobin interactions with the cytoplasmic domains of the desmosomal cadherins and found that the desmosomal cadherins regulate plakoglobin metabolic stability (Kowalczyk et al., 1994) but do not mediate homophilic adhesion (Kowalczyk et al., 1996). To test the ability of the desmoplakin amino-terminal domain to interact with the desmosomal cadherin–plakoglobin complex, we established a series of L-cell lines expressing the desmosomal cadherins in the presence or absence of a desmoplakin amino-terminal polypeptide (DP-NTP).¹ The results indicate that one important function of the desmoplakin amino-terminal domain is to cluster desmosomal cadherin–plakoglobin complexes. In addition, DP-NTP and plakoglobin were found to form complexes that could be co-immunoprecipitated from L-cell lysates. Using the yeast two hybrid system, DP-NTP was found to bind directly to plakoglobin but not Dsg1. These data suggest that plakoglobin couples the amino-terminal domain of desmoplakin to the desmosomal cadherins and that desmoplakin plays an important role in organizing the desmosomal cadherin–plakoglobin complex into discrete plasma membrane domains.