Accessorizing and anchoring the LINC complex for multifunctionality

A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell’s rear. This motif represents a new paradigm for what determines directional cell migration.Directed cell movement is instrumental for organismal development, immune responses, and the progression of diseases, such as cancer (Gardel et al., 2010). To achieve directed movement, an individual cell must establish front-to-back polarity, where there is coordinated protrusion of its front and retraction of its back (Fig. 1 A; Vicente-Manzanares et al., 2007). How polymerizing actin filaments drive protrusion of the front is understood in exquisite detail (Pollard and Borisy, 2003; Pollard, 2007). The mechanisms defining how actin filament contraction defines the back of the cell (Yam et al., 2007) have been more difficult to elucidate. Contraction of actin filaments in crawling cells is driven by nonmuscle myosin II (NMII; Vicente-Manzanares et al., 2009). NMII has three isoforms, NMII-A, NMII-B, and NMII-C, all of which can bind and contract actin filaments to generate force. Importantly, NMII-A and NMII-B have different cellular localizations, which could drive their functions (Kolega, 1998; Vicente-Manzanares et al., 2007). NMII-A localizes primarily to the front, protrusive edge and is required for adhesion maturation. In contrast, NMII-B localizes behind NMII-A, primarily to large and stable actin stress fibers in the middle and back of the cell (Kolega, 1998; Vicente-Manzanares et al., 2007). NMII-B is required for front-to-back polarity, as cells lacking NMII-B lose large stress fibers and fail to define their rear (Vicente-Manzanares et al., 2007). The major question of what drives the polarized localization of NMII-B is unknown. In this issue, Juanes-Garcia et al. report that a short serine-rich motif in NMII-B is responsible for both its localization and the establishment of front-to-back cellular polarity.Open in a separate windowFigure 1.Nonmuscle myosins in cell migration. (A) Schematic showing a top view of a crawling cell. The front of the cell is protruding (top arrow), and the back of the cell is retracting (bottom arrow). The protrusion of the edge is driven by polymerization of actin filament networks in the lamellipodium (gray hash marks). NMII-containing stress fibers (SF, dark blue lines) are assembled behind the lamellipodium. SFs are connected to focal adhesions (gray ovals) either directly or indirectly through non-NMII–containing actin bundles (Dorsal SF, light blue lines; Naumanen et al., 2008). Moving away from the cell’s front, there is a decreasing and increasing gradient of NMII-A (red wedge) and NMII-B (green wedge), respectively (Kolega, 1998). (B) Schematic of NMII-A and NMII-B isoforms. A single NMII molecule is a hexamer of two heavy chains (i.e., NMII-A, NMII-B, or NMIIC), two regulatory light chains, and two essential light chains (Vicente-Manzanares et al., 2009). The overall structure of NMII-A and NMII-B molecules is similar, with two motor domains, a coiled-coil rod domain, and a short nonhelical tail domain. The serine-rich motif is unique to NMII-B, and the role for this motif in SF contraction and the ability of the cell to apply forces to its environment are yet to be determined.Though NMII-A and NMII-B are genetically and structurally very similar, Juanes-Garcia et al. (2015) identified a serine-rich sequence (SFSSSRS) in the C terminus of NMII-B (Fig. 1 B). The authors effectively used cells depleted of NMII-B (Vicente-Manzanares et al., 2007) to test the role of this serine motif in front-to-back polarity. Although expression of wild-type NMII-B rescued front-to-back polarity, expressing NMII-B lacking the serine motif did not. Interestingly, simply inserting the serine-rich motif from NMII-B into NMII-A (NMII-A5S) conferred the ability to rescue front-to-back polarity. In addition, NMII-A5S did not localize to the front of the cell or play a role in adhesion maturation like wild-type NMII-A. Mass spectrometry analysis revealed three of the residues in the serine motif of NMII-B were phosphorylated in cells, and one of these, serine 1935, was found to be crucial for the wild-type kinetics and localization of NMII-B. A phosphomimetic point mutation, S1935D, failed to rescue front-to-back polarity in NMII-B–depleted cells. In contrast, expression of the nonphosphorylatable mutant, S1935A, localized normally to large actin stress fibers and did rescue front-to-back polarity.To provide further evidence that serine 1935 is a regulatory element of front-to-back polarity, Juanes-Garcia et al. (2015) investigated the role of PKC, which acts upstream of NMII in cell polarization (Gomes et al., 2005; Even-Faitelson and Ravid, 2006), in NMII-B–generated stable actin bundles. Cells expressing constitutively active PKC produced isotropic protrusions at the perimeter of the cell, while also failing to produce large, stable NMII-B decorated actin bundles. This isotropic protrusive phenotype was blocked when nonphosphorylatable NMII-B S1935A was expressed in cells but not with wild type or S1935D. Thus, PKC was implicated as the likely upstream regulator of NMII-B activity, which negatively regulates stable actin stress fiber formation by phosphorylating NMII-B at serine 1935. Taken together, the data presented strongly suggest that a small regulatory motif on NMII-B controls cellular front-to-back polarity in migrating cells.The findings presented in this issue by Juanes-Garcia et al. (2015) shine a bright spotlight on a family of motors that has already taken “center stage” in cellular research (Vicente-Manzanares et al., 2009). Some exciting new questions as to how NMII-B functions in the establishment of asymmetric cellular shape and function can now be addressed, including but clearly not limited to: How does the unique enzymatic activity of NMII-B’s motor domain synergize with the serine motif to drive front-to-back polarity (Billington et al., 2013)? What are the structural and dynamic implications for homo- and/or hetero-NMII filament formation (Ricketson et al., 2010; Beach et al., 2014; Shutova et al., 2014)? Does the NMII-B serine motif play a role in the establishment of more complex 3D cellular shapes? Is the serine motif required for directional cell migration through physiological environments?     

Molecular Determinants of the Coupling between STIM1 and Orai Channels: DIFFERENTIAL ACTIVATION OF Orai1�C3 CHANNELS BY A STIM1 COILED-COIL MUTANT*

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca²⁺ release-activated Ca²⁺ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by ∼50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca²⁺ entry is a key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel (2–6). However, its molecular components have remained elusive until 4 years ago; the STIM1 (stromal interacting molecule 1) (7, 8) and later on Orai1 (9–11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca²⁺ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12, 13). Its N terminus is located in the ER lumen and contains an EF-hand Ca²⁺-binding motif, which senses the ER Ca²⁺ level, and a sterile α-motif, which is suggested to mediate homomeric STIM1 aggregation (14–16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14, 17, 18). Three recent studies have independently identified the ezrin-radixin-moesin domain as the essential Orai activating domain, named SOAR (STIM1 Orai-activating region) (20) which represents so far the shortest active fragment, OASF (Orai-activating small fragment) (21) or CAD (CRAC-activating domain) (22), which includes the second, more C terminally located coiled-coil domain and the following ∼55 amino acids. The latter amino acids are suggested to contain an additional cytosolic homomerization domain indispensable for OASF homomerization and Orai activation (21).The Orai family includes three highly Ca²⁺-selective ion channels (Orai1–3) that locate to the plasma membrane, and each protein contains four predicted transmembrane segments with cytosolic N and C termini (10). All three Orai proteins possess a conserved putative coiled-coil domain in the C terminus (23, 24), whereas only the N terminus of Orai1 consists of a proline/arginine-rich region (25). Orai1 has been assumed to act in concert with STIM1 (10, 27)-activating inward Ca²⁺ currents after store depletion. The two other members of the Orai family, Orai2 and Orai3, display similar but smaller store-operated inward Ca²⁺ currents when co-expressed with STIM1 with distinct inactivation profiles, permeability properties, and 2-aminoethoxydiphenyl borate sensitivity (28–32). Recently, we have provided evidence for a store depletion-induced, dynamic coupling of STIM1 to Orai1 that involves the putative coiled-coil domain in the C terminus of Orai1 (33). Furthermore, the C terminus of STIM1, in particular the essential cytosolic region 344–442 as narrowed down by SOAR, OASF, and CAD (20–22), has been established as the key fragment for CRAC as well as Orai1 activation, because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18, 32, 33). These fragments SOAR, OASF, and CAD when co-expressed with Orai1 (20–22) exhibit enhanced plasma membrane localization in comparison with the complete STIM1 C terminus in the presence of Orai1. Specificity of interaction of SOAR to the Orai1 C terminus has been shown by its disruption (20) employing the Orai1 L273S mutant (33). Park et al. (22) have provided additional, conclusive evidence for a direct binding by combining multiple biochemical approaches demonstrating CAD interaction with Orai1.This study focused specifically on the role of the putative coiled-coil domains of STIM1 as well as Orai proteins in their coupling. Coiled-coils generally function as protein-protein interaction sites with the ability of dynamic protein assembly and disassembly (35–37). We suggest the C-terminal, putative coiled-coil domains in all three Orai proteins and the second coiled-coil motif of STIM1 as essential for STIM1/Orai communication. Moreover, the single point coiled-coil STIM1 L373S mutant allowed for differential activation of Orai channels partially stimulating Orai2 as well as Orai3 but not Orai1.     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Insect Virus Proteins (FALPE and p10) Self-Associate To Form Filaments in Infected Cells

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.Cytoskeletal elements have previously been demonstrated to be involved in several aspects of virus assembly (39, 66). For example, vaccinia virus has been shown to associate with actin during its release from the plasma membrane (15), while adenovirus is transported through the cytoplasm to the nucleus through its interaction with microtubules (17, 38). Actin has been implicated in the transport of baculovirus nucleocapsids to the nucleus (10). Other viruses contain actin in their envelopes along with viral surface glycoproteins, implying some role in the budding process (34, 54, 58). In addition, cytochalasin D, a disruptor of microfilaments, has been shown to impair the assembly of a number of different viruses (18, 42, 45). Most viruses use preexisting microtubule or microfilament proteins derived from host cells in these processes. However, we have recently demonstrated that insect poxviruses establish their own filament network during the later stages of infection, using a protein encoded by the viral genome (2).Entomopoxviruses (EPVs) are insect pathogens which replicate in the cytoplasm of infected cells and are members of the poxvirus family (reviewed in references 3 to 5 and 22). The genomes of these viruses consist of linear double-stranded DNA molecules which are 130 to 300 kb in length. Amsacta moorei EPV (AmEPV) can be grown in cultured insect cells and is the most studied member of this group of viruses (22–25, 27, 40, 50). AmEPV derives its name from the Indian red army worm, a larva from the Lepidoptera family and the host from which the virus was originally isolated (23, 25, 50). Baculoviruses also infect Lepidoptera larvae but instead replicate in the nuclei of their host cells (44). A number of baculoviruses have been studied, but knowledge of Autographa californica nuclear polyhedrosis virus (AcNPV), which infects a wide variety of larvae including that of the alfalfa leaf hopper, is most extensive (44). This virus is used routinely to produce recombinant proteins in insect virus expression systems (36, 44, 46, 49).A common property of EPVs and baculoviruses is the formation of large intracellular structures known as occlusion bodies which assemble during the late stages of viral infection. Virions are embedded within these occlusion bodies, and the process serves to protect the virus from the external environment. In the case of baculoviruses, the occlusion bodies are called polyhedra and are composed predominantly of a 31-kDa protein called polyhedrin (52). The occlusion bodies of EPVs are known as spheroids and consist mainly of a 110-kDa protein known as spheroidin (6, 9, 27, 55). Spheroidin and polyhedrin do not appear to exhibit sequence homology (6, 27, 52). A multilamellar envelope also appears to surround both polyhedra and spheroids and may help to stabilize these structures during assembly (2, 53).During the late phases of AmEPV and baculovirus infections, large bundles of filaments also appear to accumulate in the infected insect cells. In the case of AmEPV, these structures are present in the cytoplasm (2, 22, 23, 40), while those found in cells infected with baculoviruses reside both in the cytoplasm and in the nucleus (1, 14, 57). Baculovirus fibrils are composed primarily of a 10-kDa protein called p10 (47, 59). The p10 gene sequences from AcNPV, Orgyia pseudotsugata nuclear polyhedrosis virus (OpNPV), Bombyx mori nuclear polyhedrosis virus, Perina nuda nuclear polyhedrosis virus, Spodoptera exigua nuclear polyhedrosis virus (SeNPV), and Choristoneura fumiferana nuclear polyhedrosis virus (CfNPV) have been reported (13, 32, 35, 66–68). Although the different p10 protein sequences only exhibit 39 to 51% identity and molecules from different species cannot interact with one another, it is believed that the polypeptides must be structurally and functionally similar (61, 66). Deletion mutagenesis of AcNPV p10 has demonstrated that both the amino- and carboxy-terminal regions of this protein are necessary for the formation of filaments in the infected cell (60). Other studies have assigned an aggregation function to the amino-terminal half of p10 (63, 65), and it has been shown that this region contains a coiled-coil domain which is conserved among the different baculoviruses (66). It is tempting to speculate that p10 aggregation is the result of coiled-coil interaction, but direct evidence for this hypothesis is lacking. The precise role of the carboxy terminus of p10 is still unclear, although it has been proposed to interact with tubulin (11). Deletion of the entire p10 open reading frame (ORF) through homologous recombination produces a mutant virus which is still capable of replication both in vitro and in vivo but produces fragile polyhedra with fragmented polyhedral envelopes (26, 64, 65). The p10 protein has also been implicated in disintegration of the nuclear envelope of the host cell, and this function appears to be associated with the carboxy terminus of this protein (61, 65).Our laboratory (2) recently demonstrated that the cytoplasmic filaments, which characterize the late stages of infection by AmEPV, are composed primarily of a 156-amino-acid protein called FALPE (filament-associated late protein of EPVs). These filaments are closely associated with the spheroids and their membrane envelopes. FALPE is a phosphoprotein which migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 25/27-kDa doublet. This protein also contains an unusual proline-glutamic acid repeat region spanning 20 residues in the carboxy terminus of the polypeptide. The ultrastructure and close association of this protein with the occlusion bodies of AmEPV suggested that FALPE and p10 played analogous roles during infections by the respective viruses.This article addresses the structural and functional similarities between FALPE and p10. These two viral proteins are known to be major components of filamentous structures, but it is not known whether additional viral or cellular proteins cooperate during the polymerization process. In this report, we provide insight into the mechanisms which produce filaments in cells infected with either baculoviruses or EPVs. We demonstrate that p10 and FALPE can produce filaments in the absence of other viral gene products. Using the yeast two-hybrid system and a chemical cross-linking agent, we obtained evidence for self-association of either FALPE or p10. Finally, the polypeptide regions of FALPE and p10 which are required for self-association and subsequent filament formation are mapped.     

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.