Module 8: Cell Adhesion

What are the key processes required for multicellular development from a single fertilized egg?

• Repeated mitotic divisions to produce many cells

• Cell differentiation to create tissue-specific gene expression

• Cell signaling between cells

• Cells must associate and maintain connections during embryogenesis

• Formation of the inner cell mass forms the early embryo

• Embryo cells separate into three germ layers:

  • Endoderm

  • Ectoderm

  • Mesoderm

• These germ layers give rise to all cells and tissues in the body

• Cell connections are essential to prevent a “soup of cells” and enable organ/tissue functions

How was cell recognition and adhesion demonstrated experimentally?

• In 1907, H.V. Wilson separated sponge cells of two species using a fine mesh

• Mixed cells back together

• Cells from the same species recognized and re-associated

• Cells from different species did not associate

• Demonstrated species-specific cell adhesion and recognition

What did Johannes Holtfreter’s frog embryo experiment show about cell recognition and organization?

• Separated cells from two different germ layers in frog embryos

• When mixed, cells from similar tissues recognized and associated with each other

• Cells organized into tissue-specific lineages mimicking original embryo organization

• This demonstrates like cells recognize and adhere during embryogenesis

• This adhesion requires cell adhesion molecules (CAMs), which are transmembrane proteins

• After aggregation, cells form specialized cell junctions that:

  • Stabilize cell-to-cell interactions

  • Facilitate communication between neighboring cells

What are the key features of cell junctions in epithelial sheets, and what are the four main types of adhesion complexes on lateral surfaces?

• Epithelial cells connect along lateral surfaces to form sheets lining body cavities

• Epithelial sheets form:

  • Inner lining of digestive system

  • Outer layers of skin

• Cells have distinct apical (top) and basal (bottom) surfaces with different functions

• Basal surface anchors cells to extracellular matrix via hemidesmosomes

• Apical surface often has microvilli (e.g., intestinal lining)

• Four adhesion complexes connect lateral surfaces:

  • Tight junctions

  • Gap junctions

  • Desmosomes

  • Adherens junctions

What are tight junctions and what is their function?

• Also called zonula occludens

  • Located just below the apical surface of adjacent cells and seal off space between cells completely

• Prevent fluid and small molecule diffusion across cell layers

  • Important in the gastrointestinal tract to prevent enzyme leakage

• Made of linear arrays of occludin and claudin proteins

  • Appear as points where membranes are pinched together under electron microscopy

• Form a complete junctional band (not just a single junction)

  • Freeze fracturing (cells frozen with liquid nitrogen and then broken at weak points) reveals a web-like network of tight junction proteins

How do tight junctions affect membrane protein and molecule diffusion?

• Prevent diffusion of membrane proteins between apical and basolateral regions

• Block diffusion of molecules in extracellular space between cells

• Example: lanthanum hydroxide (electron-dense) cannot diffuse past tight junctions

• Tight junctions create a barrier limiting molecule movement from basal to apical surface

What are gap junctions and their role in cell communication?

• Link cytosol of adjacent cells directly

• Important for metabolic integration of tissue cells

• 1.5 - 2.0 nm in diameter to allow exchange of ions and small molecules (up to ~1 kDa)

  • Includes secondary messengers like cAMP and Ca²⁺

• Structure: 6 connexin protein subunits make up 1 hexagonal connexin hemichannel

  • Two connexon hemichannels from adjacent cells line up to form a gap junction channel

• Found in clusters forming gap junction-rich regions

• Hold cells together by pinching membranes together but still allow extracellular diffusion

• Electron microscopy shows donut-shaped gap junction arrays on lateral cell surfaces

What size molecules can pass through gap junctions and why is this important?

• Gap junction channels are about 2 nm in diameter

  • Allow diffusion of ions and secondary messengers like cAMP and calcium

• Enables rapid coordination of activities (e.g., cardiac and uterine muscle contractions)

• Stimulation of one cell can spread to others via cytosolic flow through gap junctions

• Experiment shows fluorescent molecule injected into one cell diffuses to neighboring cells connected by gap junctions

What are plasmodesmata and how do they function in plant cells?

• Plasmodesmata are plant cell structures similar to animal gap junctions

• Important in phloem function in flowering plants

• Phloem = system of elongated tubes transporting nutrients (e.g., sucrose) from leaves to rest of plant

• Phloem cells (sieve-tube elements) connected by modified/enlarged plasmodesmata forming sieve tube plates

• Sieve-tube elements are metabolically inactive

  • Companion cells support sieve-tube elements by providing ATP, proteins, and substances

  • Companion cells also connected by plasmodesmata to phloem cells

• TEM images show plasmodesmata channels span two cell membranes and the cell wall

• Plasmodesmata appear as donut-shaped structures in electron microscopy

What roles do gap junctions (plasmodesmata) play in plants?

• Phloem acts like a circulatory system, carrying sucrose from source cells (photosynthetic leaf cells) to the rest of the plant

• Green fluorescent protein synthesized in companion cells can move within the phloem via plasmodesmata

• Plasmodesmata also traffic informational macromolecules like transcription factors, gene transcripts, and small RNAs

• Viral pathogens exploit plasmodesmata to facilitate intercellular viral spread

What are anchoring junctions and how do they relate to the cytoskeleton?

• Anchoring junctions include: adherens junctions, desmosomes, and hemidesmosomes

• Distinguished by association with the cytoskeleton, especially actin filaments

• Desmosomes connect two cells

• Hemidesmosomes connect cells to the extracellular matrix

• Adherens junctions indirectly link the actin cytoskeleton between neighboring cells

What are the major families of cell adhesion molecules (CAMs) in adherens junctions and how do their interactions differ?

• Four major CAM families: cadherins, Ig-superfamily, integrins, and selectins

• Homophilic interactions (same molecule on both cells): cadherins and Ig-superfamily CAMS

• Heterophilic interactions (different molecules on cells): integrins and selectins

What are cadherins and what is their role in adherens junctions?

• Calcium-dependent cell adhesion molecules (CAMs)

• Mediate homophilic interactions (same cadherin on both cells)

• Three major classes:

  • E-cadherin (epithelial)

  • N-cadherin (neural)

  • P-cadherin (placental)

• Located near the apical surface, just below tight junctions in epithelial cells

• Adhesion involves transmembrane cadherins + cytosolic cofactors called catenins that link cadherins to the actin cytoskeleton

• Epithelial cells without E-cadherin (mediates Ca2+ dependent adhesion) gene do not aggregate in culture

• Introducing E-cadherin gene induces cell aggregation

  • Adhesion is calcium-dependent; without calcium, aggregation does not occur

How was E-cadherin’s role in tissue-specific adhesion demonstrated using GFP?

• Researchers created an E-cadherin-GFP fusion gene and introduced it into cultured cells

• Cells expressing GFP-tagged E-cadherin were mixed in calcium-containing medium

• Over time, cells expressing E-cadherin aggregated and adhered together

• GFP fluorescence accumulated at contact surfaces, showing formation of adherens junctions

• Cells only adhered to others expressing the same E-cadherin, demonstrating homophilic interaction

What is the role of neutrophils?

• A type of white blood cell

• Key players in the immune response

Why is transient cell adhesion important in neutrophil extravasation?

• Not all cell adhesion is permanent — some are transient (temporary)

• Examples of transient adhesion:

  • Cell migration during embryogenesis

  • Leukocyte migration in response to infection/injury

• Leukocytes (white blood cells) exit bloodstream via extravasation

  • A precise sequence of adhesive interactions needed

• Endothelial cells (line blood vessels) usually prevent leakage

  • Tight adhesion between these cells keeps blood cells inside

• In immune response:

  • Leukocytes slow down, adhere to endothelium, and exit bloodstream

  • Must form and break temporary connections with endothelial cells to reach infected/injured tissue

What are the three families of white blood cells (leukocytes), and which undergo extravasation?

• 1. Granulocytes (target pathogens)

  • Include neutrophils, eosinophils, basophils

  • Neutrophils:

    • Most abundant granulocyte

    • First responders to bacterial infections & trauma

    • Undergo extravasation

  • Eosinophils & basophils do not extravasate

• 2. Monocytes

  • Differentiate into macrophages

  • Perform phagocytosis (engulf bacteria, debris)

  • Can undergo extravasation

• 3. Lymphocytes

  • Include NK (natural killer) cells, T cells, B cells

  • NK cells destroy virally infected & tumor cells

  • T/B cells produce antibodies

  • Can undergo extravasation

What are the 5 steps of neutrophil extravasation and what happens in each step?

• Triggered by infection signals

• Steps:

  1. Capture

     • Temporary binding of neutrophil to endothelial cells

     • Neutrophil slows slightly, remains in bloodstream

  2. Rolling

     • Neutrophil begins to roll along vessel wall

     • Mediated by weak interactions

  3. Slow-Rolling

     • More adhesive interactions → neutrophil movement slows further

  4. Firm Adhesion

     • Strong attachment to endothelium

     • Cell shape and function change

  5. Transmigration

     • Neutrophil moves through endothelial barrier to tissue

     • Inflammation/swelling occurs due to:

       • Plasma leakage

       • Leukocyte accumulation

What triggers the initial capture of neutrophils during extravasation?

• Infection site releases cytokines (e.g., TNF-alpha)

• Cytokines affect endothelial cells lining blood vessels

• TNF-alpha binds receptors on basal surface of endothelial cells

• Triggers release of P-selectins from secretory vesicles

• P-selectins move to apical surface of endothelial cells

• P-selectins bind to ligands (selectin-specific glycoproteins) on neutrophils

• Enables transient attachment of neutrophil to vessel wall

What happens during the rolling stage of neutrophil extravasation?

• Neutrophil slows down due to weak selectin-ligand binding

• Begins rolling along the endothelial surface

• Rolling = transient attachments and detachments

• Movement driven by blood flow, but slowed by interactions with selectins

What causes neutrophils to slow-roll during extravasation?

• Closer to infection site → more selectins (P-selectin + E-selectin) on endothelium

• Higher selectin density = more binding opportunities

• Increased interactions between selectins and neutrophil ligands

• Leads to further slowing of the neutrophil

• Neutrophil now in slow rolling phase

How does firm adhesion occur during neutrophil extravasation?

• During slow rolling, neutrophil interacts with PAF (platelet activating factor) on endothelium

• PAF binds to PAF receptor (a GPCR) on neutrophil

• Other neutrophil receptors: CXCR1, CXCR2 (also GPCRs)

• PAF binding triggers intracellular signaling

  • Changes in gene expression

  • Activation of integrin adhesion molecules

• Activated integrins bind to ICAMs (intercellular adhesion molecules) on endothelium

• Result: Firm adhesion (tight binding) to endothelial surface

How do integrins contribute to firm adhesion during neutrophil extravasation?

• Inactive integrin = folded conformation (propeller & β-A domains bent down)

  • Can't bind ligands (e.g. ICAMs)

• PAF signaling triggers conformational change

  • Activates integrins (ligand-binding domain exposed)

• Active integrins bind ICAMs on endothelial cells

  • Much stronger adhesion than selectins

• Slows neutrophils further, establishing firm adhesion

• Triggers actin cytoskeleton reorganization

  • Prepares neutrophil for migration

What occurs during the final stage of neutrophil extravasation: transmigration?

• Neutrophil crawls between endothelial cells

• Produces enzymes to break endothelial cell junctions

• Involves shape changes to squeeze through gaps

• Allows movement from blood vessel to infection site

• Confocal image shows:

  • Neutrophil (red) actively migrating

  • Endothelial cells (green)

What is the sequence of neutrophil activation and cell adhesion molecule involvement in extravasation?

• Five stages: Capture → Rolling → Slow rolling → Firm adhesion → Transmigration

• Selectins:

  • Mediate early stages (capture, rolling, slow rolling)

• Integrins:

  • Activated by PAF/GPCR signals during slow rolling

  • Mediate firm adhesion

  • Enable transmigration

• Activation of cell adhesion molecules is sequential and regulated

How does the animation summarize neutrophil extravasation and activation?

• Red blood cells flow freely; neutrophils roll on endothelium

• Rolling due to PSGL-1 on neutrophil binding P-selectin

• P-selectin expression increases at infection site → slows neutrophil

• PAF binds GPCR on neutrophil → triggers integrin activation

• Activated integrins bind ICAMs on endothelial cells → firm adhesion

• Neutrophil:

  • Secretes proteases to break endothelial junctions

  • Reorganizes cytoskeleton for migration

Applied Lecture

Anti-adhesion in infectious disease treatment

What is the main cause of UTIs and how common are they?

• Uropathogenic E. coli (UPEC) is the main cause

• ~150 million cases of UTIs annually

• Can cause inflammation and long-term damage despite treatments

How do UPEC bacteria cause UTIs?

• Use pili and adhesins to adhere to epithelium

• More common in females

Steps:

• Invade urethra and bladder

• Replicate

• Immune system suppression

• Bacteria form biofilms

• Damage epithelium

• Can ascend up ureters to kidneys and renal arteries → bacteremia or septicemia

What is required for UPEC to initiate infection and what follows?

• Adhesion via pili prevents removal by urine flow/immune defenses

• Steps: attachment → invasion → replication → host cell damage

• Secrete toxins → cell death and exfoliation

Why is bacterial adhesion critical in UTIs?

• Allows bacteria to stay, multiply, and cause damage

• Initial non-covalent binding weak → becomes stronger over time as more interactions occur

What role do lectins play in adhesion?

• Lectins = proteins with sugar-binding specificity

• Example: P-selectin binds sugar residues on glycoproteins

Why is glycosylation important in host cells?

• Occurs in Golgi

• Essential for:

  • Protein folding

  • Stability

  • Function

  • Cell-cell interactions

How do UPEC pili interact with host cells?

• Pili/fimbriae (hair-like projections) have lectin proteins at their tips

• Bind to mannose-containing glycoproteins on urinary tract epithelium

What is FimH and what does it bind?

• FimD = anchor

• FimA = amino acids that create the length

• FimF + FimG = connector domains

• FimH = adhesin + lectin domain

  • Located at tip of Type 1 pili (fimbriae)

  • Binds mannose (oligosaccharide) on host cell surface

What are the domains of FimH and their functions?

• C-terminal (pilin domain): connected to pili

• N-terminal (adhesin domain): contains a carbohydrate binding pocket and binds D-mannose

• Experiment needed to test if FimH alone enables adherence

What happens in a ΔfimH mutant strain?

• Cannot form pili → no infection

• Shows FimH is necessary for pili assembly

• Is FimH alone sufficient for adhesion/internalization? → needs testing

Is FimH sufficient for adherence and internalization?

• Compare UPEC vs. FimH-coated latex beads

• Visualized with electron microscopy

• FimH enables adherence & internalization

• Key in persistent & recurrent UTIs

How does Single Cell Force Spectroscopy (SCFS) work?

• Attach UPEC cell to AFM (atomic force microscopy) probe

• Lower to contact target surface

• Measure interaction forces between cell & host

What is the Rosette Formation Assay?

• RBCs engineered to express mannose

• Mixed with UPEC in well

• High-affinity mannose binding→ diffuse rosette of RBCs

• Low-affinity mannose binding → tight pellet of RBCs due to less adherence

• Used to compare adhesion strength

How are UTIs commonly treated and what does it cost?

• Common treatment: antibiotics

• ~11 million U.S. cases/year

• ~$5 billion annually

What are the main problems with antibiotic use for UTIs?

• Antibiotic resistance → relapsing infections

• Harms gut microbiome

• Rising concern for multi-drug resistance

What happens if bacterial adhesion is blocked?

• Bacteria washed out by urine flow

• No attachment = no infection

• Strategy: interrupt adhesion step

Can soap be used to disrupt adhesion in the urinary tract?

• Soap disrupts adhesion externally

• Not usable internally in urinary tract

• Need safer, targeted alternatives

How do mannose analogs work as anti-adhesion drugs?

• Mannose + competitive inhibitors bind FimH

• Blocks E. coli binding

• Prevents adhesion to host cells

Why is FimH a strong target for drug design?

• X-ray structures available

• Enables rational drug design

• Inhibitors = low molecular weight + high potency

How are FimH inhibitors tested?

• Coat plates with mannose

• Incubate UPEC with inhibitors

• Add to coated wells

• Best inhibitors prevent bacterial binding

How do cranberries reduce UPEC adhesion?

• Contain proanthocyanidins

• Bind FimH → reduce adhesion

• Graph: Dose-dependent effect shown by AFM

  • UPEC adherence decreases with increasing concentrations of proanthocyanidins

What is the evidence that cranberry extract works?

• Non-treated: ~7 bacteria/cell

• Treated: ~2 bacteria/cell

• Confirmed with brightfield microscopy

What are the challenges in blocking bacterial adhesion?

• Hard to block all pili on E. coli

• Bacteria have multiple adhesins

• FimH inhibition alone may be insufficient

• Future: adhesion inhibitor cocktails + antibiotics