Journal of Cell Biology

Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the {gamma}-tubulin ring complex ({gamma}-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the {gamma}-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and {gamma}-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate {gamma}-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on {gamma}-TuRC activation, which occurs through several redundant pathways.

Lysosomal membrane damage triggers a multi-stage repair response essential for cellular homeostasis. Here we identify the oxysterol-binding protein-related protein ORP3 as a critical mediator of late-stage lysosomal membrane repair. Following lysosomal damage induced by L-leucine-leucine methyl ester (LLOME) or cationic amphiphilic drugs (CADs), ORP3 is phosphorylated and recruited to ER-lysophagosome contact sites via a signaling cascade initiated by lysosomal membrane ubiquitination, TAK1, p38 MAPK, and, to a lesser extent, IKK. p38-dependent phosphorylation promotes direct interaction between ORP3 and LC3B, which together with PI(4,5)P2 binding, is required for autophagic lysosome recruitment. ORP3 depletion impairs late-stage lysosomal recovery, elevates lysosomal lipid peroxidation, and reduces cell survival. A lipid transfer-deficient ORP3 mutant fails to restore lysosome function despite normal recruitment, indicating that ER-to-lysophagosome transfer of phosphatidylcholine by ORP3 is functionally required. ORP3 activity is subsequently terminated by VCP/p97-mediated deubiquitination of lysosomes. These findings define ORP3 as a MAPK regulated lipid transfer protein during the late autophagic phase of the endolysosomal damage response. SummaryLysosomal membrane damage triggers ubiquitination that activates a TAK1-p38 signaling cascade, phosphorylating the lipid transfer protein ORP3 and recruiting it to damaged lysosomes via LC3B interaction. ORP3-mediated phosphatidylcholine transfer from the ER is essential for late-stage lysosomal repair and cell survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/731146v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@49f300org.highwire.dtl.DTLVardef@f0a90dorg.highwire.dtl.DTLVardef@1eaa560org.highwire.dtl.DTLVardef@f4de4_HPS_FORMAT_FIGEXP M_FIG C_FIG

During cell migration, branched actin filaments nucleated by the Arp2/3 complex induce leading edge protrusions, whereas directionality of cell migration is controlled by microtubules. We showed previously that Adenomatous Polyposis Coli (APC) initiates branched actin assembly at microtubule tips, which can explain how microtubules control directional protrusion. Here, we investigate a link from APC to Arp2/3 complex activity. We show that protrusion-generating activity of APC resides in its N-terminal region containing the Armadillo Repeat Domain (ARD). Furthermore, Asef1/ARHGEF4, a Cdc42 GEF known to be activated by the ARD of APC, as well as Cdc42 itself and its effector N-WASP, an Arp2/3 complex activator, are all required for the assembly of branched actin filaments in neuronal growth cones and neurite outgrowth. As downregulation of Asef1, Cdc42 or N-WASP produces phenotypes similar to APC knockdown, we propose that microtubules regulate directional cell migration and neuron navigation by inducing local membrane protrusion through the APC - Asef1 - Cdc42 - N-WASP - Arp2/3 pathway. These findings bridge a significant gap in our knowledge of cell migration.

Centrosomes organize microtubules, coordinate ciliogenesis, and support accurate cell division. At the mature mother centriole, distal and subdistal appendages confer specialized functions in ciliary docking, microtubule organization, and intracellular trafficking. Nuclear distribution element 1 (NDE1) is a centrosome-associated regulator of dynein-dependent processes and ciliogenesis, but its nanoscale organization and contribution to centrosome architecture remain incompletely understood. Here, using expansion microscopy and STED super-resolution imaging, we show that endogenous NDE1 forms a ring-like structure at the subdistal appendages in mouse embryonic fibroblasts and human RPE-1 cells. NDE1 occupies an intermediate radial position between the more centriole-proximal CEP128 layer and the more peripheral ninein layer. Depletion of ODF2 or CEP128 reduces centrosomal NDE1, whereas CEP170 depletion has little effect, placing NDE1 within an ODF2- and CEP128-dependent branch of the subdistal appendage organization network. NDE1 depletion compromises centrosome integrity, reduces the centrosomal enrichment of core centriolar proteins, increases the separation between paired centrioles, and generates ectopic foci containing multiple centriolar markers. Loss of NDE1 also disrupts pericentriolar material organization and impairs the establishment of focused, centrosome-associated microtubule arrays. Furthermore, NDE1 depletion increases LC3B- and p62-positive structures and reduces autophagic flux. Together, our findings establish NDE1 as a subdistal appendage-associated factor that supports centrosome architecture and microtubule-organizing activity. More broadly, they support an emerging view of subdistal appendages as a molecularly layered platform in which distinct but cooperating components connect mother centriole maturation to microtubule organization, ciliary regulation, and intracellular trafficking.

Rab GTPase cascades drive endosomal membrane maturation by sequentially activating and inactivating Rab proteins. These transitions in Rab signaling require the coordinated actions of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The yeast VINE complex is an endosomal VPS9-family GEF that stimulates a GAP to inactivate the Rab5 homolog Vps21, suggesting a role for VINE in coordinating Rab transitions. Here we report that VINE acts through its catalytic GEF domain to promote signaling by the Rab5-related GTPase Ypt10 and establish a pool of Ypt10 at late endosomes. Ypt10 activation occurs downstream of Vps21 activity, placing Ypt10 within a late endosomal Rab cascade. Genome-wide protein proximity screens revealed a VINE-dependent interaction between Ypt10 and the GEF Mon1-Ccz1. Our data suggest that VINE and Ypt10 regulate late endosomal recruitment of Mon1-Ccz1 to enhance the activation of its substrate, the Rab7 homolog Ypt7. Together, these findings define a Vps21-VINE-Ypt10 regulatory module that adds a layer of control within the late endosomal Vps21-to-Ypt7 cascade and establish VINE as a dual Rab regulator. Through opposing activities on Vps21 and Ypt10, VINE may couple Rab5 inactivation to Mon1-Ccz1 recruitment to provide more precise control of degradative protein traffic to the vacuole. Significance statementFour Rab5-family GTPases direct protein sorting and membrane maturation in the yeast endolysosomal system, yet their individual functions, and the role of the little-studied Rab Ypt10, are unclear. Using genome-wide proximity screens, we find that the GEF complex VINE establishes a pool of Ypt10 at late endosomes downstream of Vps21, where Ypt10 recruits Mon1-Ccz1, the activator of the Rab7 homolog Ypt7. Because VINE also drives GAP-mediated suppression of Vps21, our results suggest it acts as a dual Rab regulator, coupling Vps21 inactivation to Ypt10 activation to fine-tune the endosomal Rab cascade.

Chromosome-bound RCC1 generates Ran-GTP signals to organize functional spindles for faithful chromosome segregation during mitosis and meiosis. RCC1 is the sole guanine nucleotide exchange factor (GEF) for Ran and is essential for spindle assembly during early, but not late, embryonic divisions. However, how RCC1 organizes the specialized embryonic spindle and when its function changes during early embryogenesis remain unclear. Here, using time-resolved RCC1 depletion and depletion-rescue experiments in medaka embryos, we show that RCC1 GEF activity is specifically required before the blastula stage to organize a specialized metaphase spindle mid-plane that ensures faithful chromosome segregation. Mechanistically, RCC1 promotes the accumulation of the canonical Ran effectors HURP and KIFC1/HSET, and unexpectedly, the microtubule motor dynein at the spindle midplane during early embryonic divisions. Intriguingly, a five-fold increase in RCC1 expression phenocopies RCC1 depletion, disrupting spindle-midplane organization and the accumulation of KIFC1 and dynein in a GEF activity-dependent manner. Together, our findings demonstrate that both insufficient and excessive RCC1 GEF activity compromise embryonic spindle assembly, revealing that balanced Ran activation is required to organize the specialized spindle midplane during vertebrate cleavage divisions. HighlightsRCC1 requirement changes with embryonic spindle remodeling before the blastula stage. RCC1 GEF activity is required to organize the specialized embryonic spindle midplane. RCC1 promotes the accumulation of HURP, KIFC1, and dynein at the spindle midplane. Both insufficient and excessive RCC1 GEF activity disrupt the spindle midplane organization.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

Centrioles are highly organised microtubular scaffolds which grow and mature progressively during successive cell cycles. Their molecular organisation is extensively characterized, yet the contribution of several components to centriole assembly, maturation or stability is incompletely understood. Here, using ultrastructure expansion microscopy and transmission electron microscopy, we show that ALMS1, the protein mutated in Alstrom syndrome, is required for proper centriole architecture. In absence of ALMS1, RPE1 cells exhibit shorter centrioles with defects in the microtubular wall, including broken or missing triplets or open B/C tubules. These structural defects arise after procentriole assembly. We show that ALMS1 loss selectively reduces the proximal region proteins CCDC77 and CEP44, leaving intact central and distal ones. ALMS1 is further required for the recruitment of the proximal CEP135 cap and the clearance of the {gamma}-tubulin/GCP2 pool present at the procentriole base. Our findings thus identify ALMS1 as a key organiser of the centriole proximal domain and required for remodelling and stabilising the proximal end of centrioles during cell cycle progression.

Macroautophagy is a conserved catabolic process that facilitates the degradation of cellular material by capturing it in double membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, selective macroautophagy is initiated by the scaffolding protein Atg11. Atg11 recruits the transmembrane protein Atg9, which resides in small vesicles, to autophagic cargo. Atg9 vesicles then fuse, forming the initial membrane sheet that expands into the autophagosomal membrane. While it is known that Atg9 interacts with Atg11 via a set of hydrophobic amino acids in the disordered N-terminus of Atg9, it is unclear how Atg11 mediates this interaction. To gain insight into this unknown aspect of autophagy initiation we utilized a combination of biochemical, structural, and cellular approaches. We demonstrate that the N-terminal domain (NTD) of Atg11 is the primary interaction site for Atg9, but the NTD requires clustering by the C-terminal region of Atg11 for its complete interaction with Atg9. We investigated the structure of the Atg11-NTD using cryo-EM which, in combination with AlphaFold modeling, revealed a positively charged binding pocket within the Atg11-NTD that is essential for Atg9 binding. Mutation of this conserved binding pocket leads to a loss of Atg9 binding in yeast and a reduction in the selective autophagy of mitochondria. Taken together, our results demonstrate the mechanism by which Atg11 recruits Atg9 to autophagy initiation sites.

Nuclear pore complexes (NPCs) control molecular exchange across the nuclear envelope, but how they tailor their selective permeability to meet the needs of specific cell types and/or environments remains poorly understood. We demonstrate that the strength of the NPC diffusion barrier differs across cell types, is particularly stringent in cultured neurons, and correlates with the O-linked N-acetylglucosamine (GlcNAc) modification of nucleoporins. Using conditional tools that specifically control nucleoporin GlcNAcylation, we show that GlcNAc modulates NPC permeability. Interestingly, nucleoporin GlcNAcylation is mechanosensitive, increasing in cells plated on stiff substrates, a condition where nuclear pores dilate. Indeed, we demonstrate that increasing or decreasing GlcNAcylation dilates and constricts NPCs, respectively. Further, O-linked N-acetylglucosamine transferase is recruited to modify NPCs upon their acute constriction during osmotic shock. Thus, cells employ GlcNAc to modulate steady-state NPC permeability in response to mechanical inputs and to counteract critical changes to their osmotic environment.

Microtubules (MTs) serve as intracellular tracks that enable molecular motors to transport cargos to specific cellular destinations. It has been proposed that the signals directing motor-driven transport are encoded on MTs through different isotypes, lattice conformations, and post- translational modifications (PTMs) of tubulin, or MT-associated proteins (MAPs) that decorate the MT surface. However, molecular predictions of these models have not been rigorously tested in vitro. Using isotypically pure recombinant tubulin and biochemical reconstitution, we examined how tubulin PTMs and MT lattice spacing influence MAP binding and kinesin-1 motility. We found that kinesin-1 is largely insensitive to tubulin PTMs but is strongly regulated by MT lattice spacing. Likewise, the MAPs tau, MAP7, MAP4, DCX, and MAP9 exhibit little sensitivity to tubulin PTMs, whereas the MT-binding affinities of tau, DCX, and MAP7 depend on lattice spacing. In the presence of activating (MAP7) and inhibitory (tau) MAPs, lattice spacing determines MAP occupancy and thereby controls kinesin-1 motility. These findings support a two-layer transport code in which MT lattice spacing directs MAP binding, and MAPs determine which motors can move along individual MT tracks.

Cells use multiple protein quality control (PQC) mechanisms to counteract the toxic effects of protein aggregation caused by cellular stress, ageing or disease. While it is known that PQC mechanisms differ between cellular compartments, studying such differences has remained challenging. Previously, we developed an assay to study cytosolic PQC using aggregates formed through chemically-induced dimerization (termed PIMs, particles induced by multimerization). Here, we introduce nuclear PIMs as a tool to study nuclear quality control. Using high-resolution and high-throughput imaging, we show that nuclear aggregate removal depends on the proteasome and the unfoldase VCP, but not on Hsp70. Strikingly, following dissolution many PIM subunits were exported to the cytosol via exportin-1-dependent shuttling, indicating that disaggregation and resolubilization dominated over degradation. Proteasomal disaggregation was confirmed using live-cell turnover experiments. Together, these findings reveal a mechanism in which the proteasome and VCP disaggregate, rather than degrade, nuclear protein aggregates, with the resulting subunits subsequently cleared via cytosolic aggrephagy.

Microtubule dynamic instability, driven by GTP turnover, allows microtubules in cells to re-organise themselves adaptively. In some models of dynamic instability, GTP-tubulin is selectively captured at the tips of microtubules. In others, GTP- and GDP-tubulin are both captured, but GTP-tubulin is selectively retained. To investigate, we mutated the interprotofilament interface in human 1b{beta}3 and 1b{beta}4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the 1b{beta}3 M-loop or its binding pocket into 1b{beta}4b tubulin creates tubulins that assemble in 1 mM GDP. In co-assembly experiments in GTP, such hyper-assembler mutants can recruit hypo-assembler mutants into a mosaic lattice, under conditions in which the hypo-assembler alone does not polymerise. We propose that GTP- and GDP-tubulins are captured equivalently at the tips of microtubules, but then differentially retained, based on their differing abilities to form stable interprotofilament bonds. This biased retention mechanism allows mosaic lattices to be built and dynamic instability to be tuned.

Angstrom-scale changes in microtubule (MT) lattice spacing regulate the selective recruitment of MT-associated proteins, yet how these structural states operate in cells remains poorly understood. Here, we show that MT lattice expansion, induced by protein-based expanders or microtubule-stabilizing agents such as Taxol and epothilone D, drives the relocalization of compact lattice-binding proteins, including tau, doublecortin (DCX), and the C1 domain-containing signaling protein GEF-H1, into highly curved MT-associated domains, whereas the compaction-inducing agent laulimalide suppresses this response. In contrast, the tumor suppressor RASSF1A preferentially associates with expanded lattice states, revealing differential lattice sensitivity among closely related C1 domain-containing proteins. These short, curved assemblies are enriched at MT intersections and discrete MT segments, revealing spatially heterogeneous lattice states within individual microtubules. At substoichiometric levels, compact lattice-binding proteins behave as both MT compactors and curvature sensors. Changes in osmotic pressure selectively promote dissociation of compact lattice-binding proteins, whereas expanded lattice-binding proteins remain largely unaffected. Using curved filament formation as an in-cellulo readout of compact lattice regions, we identify widespread lattice-state sensitivity across diverse MT-associated and signaling proteins. Finally, we show that these principles extend to neurons, where somatic, but not axonal, tau exhibits sensitivity to lattice expansion despite the expanded lattice architecture of distal axonal microtubules, suggesting additional neuron-specific regulation of lattice accessibility. Together, our findings identify the MT lattice as a dynamic mechanochemical platform whose nanoscale structural states spatially organize protein recruitment and signaling in cells and neurons.

Microtubules are polarised polymers that assemble into highly specialised networks in a cell-specific manner. This is controlled in part by microtubule organising centres (MTOCs), which concentrate factors necessary for microtubule nucleation and the organisation of microtubule minus ends. Neurons rely on oppositely polarised microtubule networks, with axons containing mostly plus-end-out microtubules, and dendrites contain many minus-end-out microtubules. How minus-end-out microtubule polarity is established in dendrites remains an important question. Here, we identify a new type of MTOC within the dendrites of Drosophila class I dendritic arborisation neurons, a common model for the neuronal cytoskeleton. We show that membrane swellings distributed intermittently along dendrite shafts, which we term "dendritic varicosities", contain the principal component of the microtubule nucleating complex and repeatedly generate microtubules whose plus ends grow back towards the soma. Varicosities located specifically in distal regions also contain MTOC proteins implicated in minus end anchoring, and this correlates with the accumulation of minus ends specifically in distal varicosities. Depletion of these MTOC proteins leads to major defects in minus end organisation, with microtubule buckles and loops deforming the neuronal membrane. Thus, dendritic varicosities are an important new type of neuronal MTOC that contribute to the generation and organisation of the minus-end-out microtubule network within dendrites.

Endocytosis internalises nutrients, regulates extracellular signals, and recycles membrane components. Clathrin polymerises into a coat that shapes the endocytic vesicle from the plasma membrane. However, the role of clathrins dynamic assembly in the endocytic process remains unclear. We show, using two-colour fluorescence recovery after photobleaching assays in yeast, that the clathrin coat turns over rapidly in the early phase of endocytosis, dependent on the auxilin Swa2 and its ATPase. In the late phase the turnover is stopped by the coat protein Sla1. Regulated clathrin turnover is critical for the timing of endocytic progression and for controlling coat size. In the absence of this dynamic regulation the endocytic coats become abnormally large, resulting in the failure of the final actin-driven vesicle budding. These findings reveal that, in addition to its classic structural function, the dynamic properties of the clathrin lattice are critical for both the temporal and mechanical aspects of endocytosis.

How growth factor receptors decode ligand identity into distinct cellular responses remains a fundamental question in cell signaling. Here, we identify a receptor-proximal mechanism that links ligand-specific EGFR activation to distinct endocytic and biological outputs. We show that EGF, but not TGF, selectively engages a RAC1-PLC{gamma}2-IP3R signaling axis that supports EGFR non-clathrin endocytosis (NCE). PLC{gamma}2, but not PLC{gamma}1, localizes to RTN3-dependent PM-ER contact sites, where it generates localized Ca{superscript 2} signals required for completion of NCE, mitochondrial activation and cell motility. This specificity requires the RAC-binding interface of PLC{gamma}2 and is associated with RAC1-dependent formation of CTxB-positive PM regions, indicating that spatial organization contributes to signaling specificity. TGF fails to efficiently assemble the EGFR-associated organelle platform and instead favors clathrin-dependent EGFR uptake, prolonged proliferative signaling, greater organoid yield, and reduced migration compared with EGF. Together, our findings identify the RAC1-PLC{gamma}2 axis as the key determinant that decodes EGFR ligand bias by coupling receptor trafficking to the metabolic program that supports cell migration.

Adherens junctions physically connect neighboring cells and are built around classical cadherins, homophilic transmembrane proteins that link to the actin cytoskeleton. Classical cadherins can organize into ordered arrays in vitro, but whether they do so in cells remains to be established. Here, we use fluorescence polarization microscopy to show that the classical cadherin N-cadherin is orientationally ordered at cardiomyocyte cell-cell junctions. Whereas the desmosomal cadherin desmoglein 2 was similarly ordered across junction types, N-cadherin order was spatially heterogeneous. Order was lowest where organized myofibrils terminate at high-load, vinculin-enriched axial junctions and highest at low-load, vinculin-poor lateral junctions. This inverse relationship between order and mechanical load suggests that robust cadherin-mediated adhesion does not require ectodomain order. Our findings provide evidence that a classical cadherin is orientationally ordered in cells and show that mechanically active adhesions adopt distinct organizational strategies according to local mechanical demands. Summary StatementAt cardiomyocyte junctions, N-cadherin is ordered where mechanical load is low but disordered where load is high, suggesting that cadherin organization adapts to local force conditions.

The polarization of motile cilia requires that the centrioles, from which cilia are formed, display rotational asymmetry. This property is manifested in the presence of asymmetrically distributed appendages and relies on evolutionary conserved mechanisms. These mechanisms are also at play in cells that form primary cilia despite the lack of ciliary motility and asymmetric centriole appendages in this context. Here, we find that a complex consisting of CCDC61, KIAA1328 (K1328), and Centlein (CNTLN) contributes to the establishment of centriole rotational asymmetry. In cells with a primary cilium, this complex is required for assembling a linker that repositions the daughter centriole close to and orthogonal to the proximal end of the mother centriole/basal body. The CCDC61/K1328/CNTLN complex also triggers the asymmetric recruitment of pericentriolar matrix components around newly assembled centrioles, which likely facilitates the later attachment of the basal body-daughter centriole linker. Overall, our results establish that rotational asymmetry relies on the coordinated recruitment of asymmetric landmarks along centrioles and is necessary for positioning the centrioles in a configuration that is widely conserved in ciliated cells.

{delta}-Catenins interact with both classical and desmosomal cadherins and play essential, yet incompletely understood, role in adherens junctions (AJs) and desmosomes. According to the prevailing model, {delta}-catenins are recruited to these junctions exclusively through direct binding to the cadherin juxtamembrane domain (JMD). Here, we show that plakophilin 4 (Pkp4), one of the AJ-associated {delta}-catenins, is recruited into AJs through two distinct and independent mechanisms. The first is the conventional pathway based on direct interaction with the cadherin JMD. The second is a previously unrecognized mechanism that targets Pkp4 specifically to lateral AJs, submicron-sized, exceptionally stable junctions located along the mid-lateral region of epithelial cell-cell contacts. This targeting occurs independently of the cadherin JMD but requires an interaction with phospholipid-rich plasma membrane domains. We identify the conserved insert between ARM repeats 5 and 6 as the phospholipid-binding module of Pkp4. Because both membrane-binding determinants within this insert, a palmitoylated cysteine residue and a polybasic motif, are highly conserved throughout the {delta}-catenin family, our findings suggest that recognition of specialized plasma membrane domains is a general property of {delta}-catenins. We propose that the interplay between cadherin- and phospholipid-dependent targeting mechanisms enables individual {delta}-catenins to selectively stabilize distinct cadherin-based cell-cell junctions, thereby contributing to the overall architecture of the cell-cell adhesion system.