Outline: Cell-Cell and Cell-ECM Interaction -Integration from cells to tissues- I. Cell-Cell Adhesion (cell Junctions). II. Cell-Matrix Adhesion. III.Components of the extracellular Matrix. Yi-Ping Hsueh 2019 Trans-Well system Epithelial cells 1. Epithelial cells 2. Cell migration 1 Cell junctions: Epithelial cells Cell junctions link to cytoskeletons Tight Junctions Tight junctions and adherens junctions are linked to the actin Freeze-fracture EM cytoskeleton, and desmosomes and hemidesmosomes are linked to intermediate filaments. a major constituent of TJ strands Claudins with molecular masses of ~23 kD comprise a multigene family consisting of 24 members in mice/humans. 2 Tight junctions: 1. To seal off body cavities 2. To restrict diffusion of membrane components. 3. To maintain blood-brain barrier. Capillary Intestinal lumen Epithelial cells Epithelial Basal lamina Current Biology Dec, 2011, Vol. 21, Featured video The composition of tight junctions. Assembly of epithelial tight junctions and regulation of proliferation and differentiation. aPKC, atypical protein kinase C; huASH1, human ZO-1: absent, small or homeotic discs 1; JAMs, junctional adhesion molecules; MAGI, •Tight junction associated Membrane-associated MAGUK guanylate kinase inverted; MUPP1, multi-PDZ domain protein 1; Pals1, Protein •In confluent epithelial associated with Lin-7 1; culture, ZO-1 is restricted PAR, Partitioning defective; PATJ, Pals1- at the junction. In associated tight junction subconfluent culture, ZO1 protein; PP2A, protein is present in the nuclei. phosphatase 2A; PTEN, phosphatase and tensin homologue; ZO, zonula •ZO-1 interacts with a Y- occludens; ZONAB, ZO-1- box transcription factor associated nucleic-acid ZONAB and regulates binding. EGFR member ErbB2, which is critical for regulation of epithelial cell Some of the components that are thought to become recruited to TJs do not localize proliferation. exclusively to TJs, but can also localize to the nucleus (for example, huASH1 and ZONAB) or to other areas of the plasma membrane (for example, PTEN, PP2A and heterotrimeric G PNAS 93:10779 (1996) proteins). In addition, many TJ components interact directly or indirectly with actin filaments. JCB 160:423 (2003) 3 claudin-11/oligodendrocyte-specific protein (OSP) Glial Cells * found in CNS myelin and testis * Tight junction strands in CNS myelin and sertoli cell are absent in Osp/claudin-11 null mice CNS: Oligodendrocytes Astrocytes * CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Optic nerve PNS: Schwann Cell Forming myelin sheath Electrical information delivery Cell 99:649 (1999). Myelin Sheath: claudin-11/oligodendrocyte-specific protein (OSP) Isolation * found in CNS myelin and testis Induce action potential to pump from node to node along axon * Tight junction strands in CNS myelin and sertoli cell are absent in faster conduction of action potential along axon Osp/claudin-11 null mice * CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Optic nerve Cell 99:649 (1999). 4 Gap Junctions: Connexins, connexons, intercellular channels and gap junctions. Connexins: * 21 human genes and 20 mouse genes for connexins have been identified. 1. Allow small molecules (< 1.2 nm in diameter or 1200 * Each connexin shows tissue- or cell-type-specific expression, and most organs Dalton) to pass between adjacent cells, e.g. amino and many cell types express more than one connexin. acids, nucleoside phosphates, cAMP and ions… * Cell coupling via gap junctions is dependent on the specific pattern of connexin 2. The channels close in the presence of very high gene expression. calcium in the cytosol. * Regulation of connexin expression is mainly controlled at transcriptional level. Charcot-Marie-Tooth Disease: Gap junctions in PNS and CNS 1. An X-linked demyelinating disorder of peripheral nervous system. 2. Caused by mutation in connexin32 (Cx32), which is expressed abundantly in Schwann cells. 3. Reduced translation of connexin-32 appears to be caused by a mutation in its IRES in the 5‘ untranslated region of the connexin-32mRNA. 4. Cx32 is concentrated in special regions of Schwann cells. 5. The gap junctions in Schwann cells allow rapid transportation of the materials from the cell body to periaxonal region. Expression of Cx32 in PNS and CNS myelinated fibers. Schematic diagram showing the localization of Cx32 and other GJ proteins in myelinating Schwann cells in the PNS (left) and in the CNS (right) oligodendrocytes (O) as well as astrocytes (A). In Schwann cells Cx32 forms GJs through the non-compact myelin areas including the paranodes and Schmidt-Lantermann incisures. Coexpressed Cx29 likely forms hemichannels in the innermost aspect of these areas adjacent to the axonal membrane and apposing voltage-gated potassium channels (VGKC). In the CNS, all oligodendrocytes express Cx47 in cell bodies and proximal processes, and Cx47 forms most O:A GJs, while both Cx47 and Cx32 form O:O GJs. In large myelinated fibers they coexpress Cx32, and in small myelinated fibers (data not shown), Cx29. Astrocytes connect with oligodendrocytes and other astrocytes mainly with Cx43 and Cx30. NF, Neurofilaments; MT, microtubule; N, node; P, paranode; J, juxtaparanode; VGNaC, voltage-gated sodium channels. (JN 31:17753, 2011) 5 Gap junctions synchronize the condition between neighboring cells. Chemical Synapse Including small molecules and ions. Gap junctions also act as electrical synapses. Coupling of SCN neurons via gap junctions is important for the precision of circadian behavior. (NN 8:61, 2004) Gap junctions also regulate oocyte maturation. Individual SCN neurons contain the molecular machinery necessary to generate circadian oscillations. One gap in our knowledge is the lack of understanding of how these single-cell oscillators are coupled. The new study demonstrates that SCN neurons are coupled through direct electrical connections. This coupling is lost in mice deficient in Cx36. Bottom, schematics of wheel-running activity records from WT and Cx36-deficient mice. Animals maintained in constant darkness show rhythms driven by the endogenous timing system. Each horizontal row represents the activity record for a 24-hour day. Successive days are plotted from top to bottom. The colored bars represent activity. The WT mice express robust circadian rhythms of locomotor activity with period shorter then 24 h. The onset of activity is typically under precise control. In contrast, the Cx36- deficient mice showed rhythms that were weaker and less coherent than controls. Without the Cx36, the circadian clock still keeps time but lacks the temporal precision that typically characterizes the behavioral output. 6 Adherens Junctions and Desmosomes: Cell junctions: Epithelial cells Cadherin-containing junctions a- and b- catenins plakoglobin Cadherin family desmocollin E-cadherin desmoglein Cadherin family: Cadherin 1. E-, P-, N-cadherins…..(more than 300 cadherins) family 2. Defined by cadherin domain 3. Ca2+-dependent cell adhesion molecules 4. Homophilic interaction. 5. particularly important during early differentiation. 7 Adherens Junctions The dual role of ß-catenin in cell adhesion and transcriptional activation. 8 Desmosome Desmosome: * They are the most prevalent adhesive structure in tissues that undergo considerable mechanical strain, including heart and skin. * Defects in desmosome-mediated cell–cell adhesion can lead to tissue Desmosome-Interfilaments linkage regulates fragility syndromes. the balance of cell forces Atomic Force Microscope * Desmosomal proteins are also required for normal embryonic development. Knockout mice for several desmosomal proteins die in utero. Depending on the protein studied, death occurs either around the time of implantation, at mid- gestation or shortly before birth. So far, it appears that structural defects leading to abnormal histo-architecture and tissue fragility are the main cause of death. * There is no evidence that loss of a desmosomal protein would abort specific cell lineages or differentiation programs. Measurement of cell stiffness Eur J Cell Biol. 2005 84:215-23. Broussard et al., MBC 28:3156, 2017 9 Desmoglein 1 (Dsg1)–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis Getsios et al., JCB June 22, 2009 Stratified epithelium, such as skin The expression of Dsg2 and Dsg3 is permissive for EGFR–Erk1/2 signaling in the basal layer, which suppresses terminal differentiation. In contrast, the induction of Dsg1 in the suprabasal layers dampens EGFR–Erk1/2 activity, allowing for the progression into a more differentiated state and expression of Dsc1, K10, and loricrin. Cell junctions: Epithelial cells 10 II. Cell-matrix adhesion Focal Adhesion Integrin-containing junctions connect cells to the substratum. Model for integrin activation Model for integrin activation Inside-out activation Inside-out activation a b Cellular stimulation induces a conformational change in talin that exposes its talin head domain. The talin head domain binds to the β cytoplasmic tail, which displaces the α tail from its complex with the β tail, which in turn leads to an unclasping and a membrane associated structural change of the cytoplasmic avb3 integrin face (Vinogradova et al. 2002, 2004). Notice the proposed shifted membrane interface for both membrane-proximal helices before and after unclasping (green bars), which suggests a fanning-out Inactive form unclasping process (Vinogradova et al. 2004). The unclasping