Mechanobiology Notes PDF

Notes Week 1: Fundamental Concepts Definition and Scope Mechanobiology studies how mechanical forces and geometry affect cell growth, motility, and functionality. It combines insights from cell biology, biochemistry, genetics, medicine, and engineering...

Notes Week 1: Fundamental Concepts Definition and Scope Mechanobiology studies how mechanical forces and geometry affect cell growth, motility, and functionality. It combines insights from cell biology, biochemistry, genetics, medicine, and engineering. Cellular Environment - Cells exist in a gel-like environment - Cells constantly probe, push, and pull their surroundings - Mechanical forces regulate cell division, with cells exerting traction forces at protrusion tips Cell Cytoskeleton - Maintains cell shape and provides support - Anchors organelles and enzymes - Enables contractility and movement - Facilitates intracellular transport - Composed of actin filaments, microtubules, and intermediate filaments Mechanotransduction Types 1. Passive Mechanosensing: External forces transduced into cells 2. Active Mechanosensing: Internal forces generated by cells to detect environmental changes Mechanisms - Through adhesion complexes - Via ion channels (mechanosensitive membrane-bound proteins) - Through integrin-mediated pathways connecting ECM to nuclei Cellular Responses to Mechanical Cues - Migration - Proliferation - Apoptosis - Differentiation - Focal contact formation - Anchorage Engineering Tools and Applications Research Tools - Cell stretching devices - Atomic Force Microscopy (AFM) - Optical tweezers - Microfluidic devices Biomaterials - Provide adhesion sites and cell factor binding sites - Control degradation - Influence mechanics through scaffold stiffness - Create various topographies for cell guidance Stem Cells and Mechanobiology - Substrate stiffness influences stem cell differentiation - Different elasticity ranges guide specific cell types: - 0.1-1 kPa: Neuronal - 8-17 kPa: Muscle - 25-40 kPa: Bone Clinical Significance Disease Connection Abnormal mechanotransduction can lead to diseases through: - Changes in cell mechanics - Alterations in ECM structure - Disrupted mechanosensation - Deregulated mechano-chemical conversion Applications - Understanding tissue regulation - Developing new treatments for arthritis, osteoporosis, and cancer - Improving medical devices - Studying diseases like asthma, malaria, and heart disease Mechanomics - Recognizes mechanics as fundamental to cell and developmental biology - Emphasizes physical cues as important as chemical signals - Highlights biomaterials' role in addressing mechanobiology challenges - Demonstrates clinical relevance in various diseases  Week 2 Major Components 1. Actin Filaments (Microfilaments) - **Structure and Formation** - Composed of globular (G-actin) monomers that polymerize into filaments (F-actin) - Polar structure with barbed and pointed ends - Semi-flexible polymers requiring ATP for assembly - Polymerization is reversible and depends on ATP hydrolysis - **Functions** - Maintain cell shape by resisting tension - Enable cell movement - Provide mechanical strength - Link transmembrane proteins to cytoplasmic proteins - Form contractile ring during cytokinesis - Interact with myosin for muscular contraction - **Actin Architecture** - Cortical networks: non-aligned networks below plasma membrane - Stress fibers: bundled filaments with myosin - Lamellipodia: branched networks for cell movement - Filopodia: aligned bundles for directional response 2. Microtubules - **Structure and Formation** - Rigid, hollow rod-like structures - Composed of α and β tubulin heterodimers - 13 protofilaments form hollow tubular structure - Dynamic structures with (+) and (-) ends - **Functions** - Maintain cell shape by resisting compression - Enable cell movement - Facilitate intracellular transport - Guide mitosis - Direct motor proteins carrying organelles - **Microtubule Organizing Centre (MTOC)** - Controls microtubule number, polarity, and assembly - Centrosome contains pair of centrioles - Important for cell division and mitotic spindle formation 3. Intermediate Filaments - **Structure and Types** - Various types including keratins, nuclear lamins, neurofilaments, vimentins - Size ranges from 40-200kDa - Non-dynamic and non-polar - Complex assembly process from monomers to rope-like filaments - **Functions** - Structural support within cells - Maintain cell shape against tension - Provide mechanical strength - Resist shear stress - Connect other filaments to nucleus Biological and Medical Implications Mechanical Signaling and Disease - **Cancer Development** - Tissue stiffness increases during cancer progression - Cytoskeleton dynamics influence tumor mass mechanics - Important for cancer metastasis and invasion - Potential target for anti-metastatic therapies - **Other Pathologies** - Atherosclerosis: mechanical tension on actin affects cell death - Alzheimer's disease: involves abnormal actin filament/ADF/cofilin rods - Affects synaptic function in neurological conditions Cell Response to Mechanical Cues - Influences cell migration, proliferation, apoptosis, and differentiation - Involves focal contacts and mechanical signaling - Cytoskeleton remodeling occurs in response to mechanical loading Extracellular Matrix Interaction - Composed of cross-linked proteins and biopolymers - Provides mechanical support for cells - Regulates biological functions - Interacts with cytoskeletal components through various mechanisms  Week 3 Fundamentals of Cell Signaling Cells communicate to maintain functional states, proliferate, differentiate, and migrate. This communication occurs through various signaling mechanisms: - Cell-cell contact - Paracrine signaling - Autocrine signaling - Endocrine signaling Cells communicate through: - Electrical signals - Chemical signals - Mechanical signals Mechanotransduction Mechanotransduction is the process where cells convert physical forces into biochemical responses, leading to changes in gene expression and cell function. This process involves four key steps: 1. Mechanocoupling 2. Biochemical coupling 3. Signal transfer from sensor to effector cells 4. Effector cell response Types of Mechanosensing - Passive mechanosensing: External forces are transduced into cells - Active mechanosensing: Cells generate internal forces to detect external environment changes Key Components and Mechanisms Integrins and Focal Adhesions - Integrins are transmembrane receptors that facilitate cell adhesion and act as mechanoreceptors - Focal adhesions connect the extracellular matrix (ECM) to actin filaments - Components include integrins, talin, and vinculin Cytoskeleton Components - Actin filaments - Microtubules - Intermediate filaments Force Transmission Pathway - Focal adhesions connect ECM to actin filaments - Cytoskeleton couples to nucleus through nesprins - Nesprins interact with inner nuclear membrane proteins (SUN1 and SUN2) - Nuclear lamins and SUN proteins bind to nuclear pore complexes Signaling Pathways Rho/ROCK Signaling - Rho family consists of small guanosine triphosphatases - Controls actin cytoskeleton regulation - Acts as molecular switch for signal transduction - RhoA and ROCK affect myosin-generated cytoskeletal tension - Influences MSC lineage commitment Hippo Signaling Pathway - Controls organ size through cellular proliferation, survival, and differentiation - Key effectors: YAP and TAZ - Responds to: - Substrate stiffness - Cell density - Stretching - Cell shape - Cellular tension Delta-Notch Pathway - Delta: Transmembrane protein on signaling cell - Notch: Receptor on receiving cell - Interaction leads to: - Notch conformation changes - Cytoplasmic domain translocation to nucleus - Transcription factor activation ECM Interactions and Mechanical Response - Matrix stiffness affects integrin expression - Soft matrix: - Impedes cell spreading - Reduces focal adhesion formation - Downregulates β1 integrin through lysosome-mediated degradation Clinical Relevance - Multiple pathways affect cell birth, differentiation, and maintenance - Pathways form complex, non-linear networks - Deregulation can lead to various diseases - Mechanical cues play crucial role in stem cell differentiation - Biomechanical signals guide cell behavior and fate - Mechanical stresses are present through external forces and endogenous cytoskeletal activity  Week 4 Core Concepts - Mechanobiology involves mechanosensing (cells sensing mechanical signals) and mechanotransduction (translating mechanical signals into cellular responses) - Mechanical forces play crucial roles in embryonic development, patterning, and organogenesis - Mechanical cues regulate cellular processes including adhesion, migration, proliferation, and differentiation Homeostasis - Represents a dynamic equilibrium rather than a constant state - Mechanical homeostasis is an active process requiring sensors and effector mechanisms - Disruption can lead to disease progression and tumor formation - Examples of disrupted homeostasis: - Breast cancer tissue becomes 10 times stiffer than healthy tissue - Liver cirrhosis affects tissue mechanics Extracellular Matrix (ECM) Structure and Components - Contains over 300 proteins, 200 glycoproteins, 30 proteoglycans, and glycosaminoglycans - Major components include: 1. Proteins - Collagen (25-35% of body proteins, over 25 types) - Elastin (can stretch up to 150% length) - Other proteins: fibronectin, vitronectin, laminin, vinculin 2. Glycoproteins - Conjugated proteins with carbohydrate components 3. Glycosaminoglycans (GAGs) - Maintain and support collagen and elastin - Aid in moisture retention 4. Proteoglycans - Combinations of proteins and GAGs - Serve as lubricants and support elements ECM Functions - Provides structural support for tissues - Anchors and supports cells - Facilitates cell signaling - Delivers biochemical and mechanical cues - Undergoes constant remodeling Key Players in Mechanical Homeostasis 1. Extracellular Matrix 2. Effectors - Fibroblasts: build and maintain ECM - Secrete proteins and proteases - Can differentiate into myofibroblasts under high tensile stress 3. Sensors - Integrins: sense and regulate ECM mechanics - Connect to actomyosin cytoskeleton - Involve proteins like ROCK, MAPK, and linker proteins ECM in Different States Normal Tissue - Maintains organized structure - Contains relaxed meshwork - Effectively resists compressive stresses - Has controlled feedback mechanisms Aged Tissue - Shows compromised junctional proteins - Develops gaps between epithelial cells - Demonstrates reduced basement membrane protein synthesis - Becomes mechanically weaker and less elastic Tumor Tissue - Loses tissue organization - Becomes stiffer than surrounding tissue - Shows increased ECM deposition and remodeling Cell-ECM Interactions - ECM stiffness influences cell behavior - Affects cell spreading, focal adhesions, and stress fibers - Impacts cell cycle progression and gene expression - Different cell types require different stiffness levels - Force and load responses - Cells actively respond to mechanical loads - Long cyclic loading leads to cytoskeletal remodeling - Mechanotransduction - Converts mechanical stimuli to biochemical information - Force transmission increases with ECM stiffness Applications and Future Directions - Important for tissue engineering and regeneration - Potential applications in: - Cancer detection and progression monitoring - Drug discovery  Week 5 Cellular Environment and Responses - Cells exist in a jelly-like environment - Cells constantly probe, push, and pull their surroundings - Biomechanical cues trigger various cellular behaviors: - Migration - Proliferation - Apoptosis - Differentiation - Focal contacts - Anchorage Role of Biomaterials ### Key Factors Affecting Cell Response - Stiffness (Young's modulus) - Topography/architecture - Binding sites - Matrix strains - Protein adsorption on surfaces Types of Biomaterials 1. Metals and Ceramics - High mechanical stability - Limited to corrosion-free metals - High elastic modulus (>100 GPa) - Difficult to functionalize 2. Polymers - Easily fabricated into various forms - Readily incorporate surface patterns - Tailorable physical properties - No ion release - Easy to functionalize 3. Hydrogels - Highly hydrated 3D polymeric networks - Can be synthetic (PAAM, PVA, PEGDA) or natural (collagen, gelatin, alginate) - Crosslinking mechanisms: - Physical: Through crystalline regions and non-covalent interactions - Chemical: Through covalent bonds between polymer chains Hydrogel Formation and Properties Crosslinking Methods - Chain-growth polymerization - Solution polymerization - Photopolymerization - Irradiation polymerization Cell Adhesion and Mechanics - RGD peptide sequence important for cell adhesion - Stiffness can be controlled through: - Macromer concentration - Degree of functionalization - Photo-assisted stiffening 2D vs 3D Culture Systems Key Differences 2D Systems: - Soluble gradients absent - Continuous matrix layer - Free migration along 2D plane - Limited adhesion 3D Systems: - Soluble gradients present - Fibrous matrix with multidirectional surfaces - Migration limited by 3D environment - Adhesion in all directions Challenges in 3D Culture - Cell incorporation methods - Oxygen availability - Nutrient distribution - Experimental analysis limitations Topographical Features Creation Methods - Dip pen nanolithography - Photolithography - Microfluidics - Focused laser light Cell Responses - Cells recognize and align with topographical features - Influence focal contact assembly - Affect stress fiber formation - Impact stem cell differentiation  Week 6 Importance of Force Microscopic Techniques in Biology - Cells respond to mechanical properties of their environment by adjusting their own mechanical properties - Key factors include contact with neighboring cells, tissue structure, and tissue tension/compression - Mechanical properties affect cell differentiation, migration, proliferation, and survival - Substrate stiffness influences cell behavior and development - Mechanical changes can indicate diseases like cancer, with cancer cells typically being softer than healthy cells Atomic Force Microscopy (AFM) Basic Principles and Operation - Invented in 1986 by Binning, Quate, and Gerber - Uses cantilever deflection to measure surface properties - Operates based on Hooke's law (F=-ks) - Multiple operation modes: - Contact mode: High resolution but potentially damaging - Tapping mode: Good for biological samples - Non-contact mode: Minimal damage but lower resolution Applications 1. Imaging Capabilities - Can image nanoscale biological samples - Provides detailed surface topography - Capable of true atomic resolution - Effective for live cell imaging 2. Cell Mechanics Analysis - Measures force curves during approach and retraction - Analyzes mechanical properties including: - Elasticity - Stiffness - Adhesion - Deformation - Dissipation 3. Cancer Research Applications - Identifies mechanical differences between cancer and normal cells - Maps nanomechanical properties across tissue samples - Shows progression from normal to cancerous tissue through stiffness measurements Traction Force Microscopy (TFM) Principles and Methodology - Measures forces generated by cells on their environment - Uses deformable substrates with embedded fluorescent beads - Forces range from piconewtons to nanonewtons - Two main approaches: 1. Polyacrylamide (PAA) gel substrates 2. Micro/nanopillar arrays Key Features - Measures cell-generated forces without external force application - Can track force changes over time - Provides insights into cell adhesion and migration - Forces vary by cell type and condition Applications - Cancer research: Different force patterns between cancer and normal cells - Drug response studies - Cell behavior analysis - Disease diagnosis and treatment development Comparison of AFM and TFM AFM Characteristics - Force range: 10⁻⁷-10⁻¹¹ N - Advantages: - High resolution - Minimal disruption - 3D surface profiling - Multiple mechanical parameter measurements - Limitations: - Requires cell adhesion - Slow scanning rate - Potential cellular response to contact TFM Characteristics - Force range: 10⁻⁷-10⁻⁹ N - Advantages: - Precise force localization - Adjustable substrate properties - Suitable for adhesion and migration studies - Limitations: - Complex substrate topology - Potential cell-substrate interactions - Cannot measure certain mechanical properties  Week 7 Optical Tweezers - Uses laser light to generate optical traps for manipulating particles - Can hold, move, rotate, join, separate and stretch particles without contact - Suitable for particles 20nm to several micrometers in size - Exerts forces of 0.1 to 100 pN - Applications include measuring single molecule bond strength and cellular force transmission Working Principle - Based on momentum transfer from photons - Forces arise from refraction and reflection of laser light - Gradient force pulls particles toward high intensity regions - Trap strength characterized by stiffness (K) - Force follows Hooke's law: F = -Kx - Uses quadrant photodiode for detecting deflections Optical Stretcher - Novel laser tool for manipulating single cells in suspension - Cells trapped between two opposing laser beams - Measures viscoelastic properties through stress-strain experiments - Uses 780nm wavelength with minimal reflection/absorption Micropipette Aspiration - Applies suction pressure to analyze cell mechanics - Measures deformation and calculates elastic modulus - Works for both suspended and substrate-attached cells - Can measure nuclear mechanics - Key parameters: suction pressure (ΔP), aspirated length (Lp), pipette radius (Rp) Applications 1. Cell mechanical properties measurement 2. Single cell manipulation 3. Tension heterogeneity mapping in tissues 4. In vitro diagnostics Comparison of Methods Optical Tweezers - Force range: 10^-11 to 10^-14 N - Advantages: Precise 3D manipulation - Limitations: Limited force level, potential heating effects Optical Stretcher - Force range: 10^-9 to 10^-11 N - Advantages: Non-contact, high throughput - Limitations: Laser power constraints Micropipette Aspiration - Force range: 10^-7 to 10^-10 N - Advantages: Real-time correlation, high accuracy - Limitations: Requires analytical models Fluorescent Proteins (FPs) in Mechanobiology Key Techniques 1. Fluorescence Resonance Energy Transfer (FRET) - Measures protein interactions at 8-10nm resolution - Used for visualizing signal transduction - Example: Src biosensor for mechanical stimulation response 2. Fluorescence Recovery After Photobleaching (FRAP) - Analyzes protein mobility and dynamics - Measures diffusion coefficients 3. Fluorescence Loss in Photobleaching (FLIP) - Similar to FRAP but analyzes outside bleached region - Assesses molecular mobility 4. Fluorescence Lifetime Imaging Microscopy (FLIM) - Measures excited state lifetime - Independent of fluorophore concentration - Applications: pH imaging, calcium imaging, drug release monitoring 5. Chromophore-assisted Light Inactivation (CALI) - Manipulates protein functions at subcellular level - Uses reactive oxygen species - Example: Light-induced killing of HeLa cells Each technique offers unique capabilities for studying cellular mechanics and protein dynamics, with applications ranging from cancer research to embryo development studies.  Week 8 Cancer Fundamentals Cancer represents a significant global health challenge characterized by abnormal cell division and growth. Unlike normal cells, cancer cells exhibit uncontrolled proliferation and can form tumors that are either benign or malignant. Cancer cells can originate from various tissues including epithelia, connective tissue, lymph nodes, and blood. Cancer Invasion and Metastasis Metastasis, responsible for approximately 90% of cancer deaths, involves cancer cells spreading from the primary tumor to surrounding tissues and distant organs. The process includes: - Carcinoma development and invasion through the basement membrane - Angiogenesis and intravasation into blood vessels - Extravasation at secondary sites - Invasion of secondary tissue Tumor Microenvironment (TME) The TME is crucial for solid malignancies and influences: - Tumor growth, proliferation, and invasion - Drug resistance and cancer recurrence - Comprises various cells including tumor cells, cancer stem cells, inflammatory cells, and cancer-associated fibroblasts Physical Properties of TME - Increased deposition of fibrillar matrix components - Enhanced ECM protein crosslinking - Elevated matrix stiffness affecting cell cytoskeletal tension - Creation of physical barriers that hinder therapeutic efficacy Mechanical Cues in Cancer Development Stiffness - Increased matrix density and stiffness observed in breast cancer patients - Cells show different behaviors on soft versus stiff substrates - Tumor cells demonstrate higher proliferation on matrix rigidity corresponding to secondary locations Topography - ECM fibers arrange in parallel anisotropic orientation during progression - Facilitates cancer cell migration - Malignant cells evade growth inhibitory cues Mechanical Forces 1. Stretching: - Results from tumor interstitial fluid pressure - Leads to mechanical stretching of tumor cortex - Increases cancer cell proliferation 2. Confinement: - Affects circulating tumor cells morphology - Impacts cell division and spindle formation 3. Shear Stress: - Experienced by cancer cells in circulation - Influences cell cycle progression through integrin activation Tumor Rigidity vs. Tumor Stress Tumor Rigidity: - Caused by elevated ECM deposition - Activates stromal cells - Induces EMT in epithelial cells - Amplifies growth-factor signaling Tumor Stress: - Results from increased cell proliferation and blood flow - Induces hypoxia - Augments cell proliferation - Increases chemoresistance Research Tools and Approaches Analytical Tools: - Atomic Force Microscopy (AFM) for analyzing tumor cell stiffness - Capability to measure stiffness variations in human cancer biopsies - Analysis of tumor cell spheroids Bioengineering Approaches: - Hydrogels as research platforms - Microfluidic devices - Organ-on-chip platforms - 3D assessment methodologies - Mathematical modeling Future Perspectives - Biophysical cues emerging as critical regulators and therapeutic targets - Focus on physical cues' effects on tumor stromal cells - Development of cell-based stiffness sensors - Understanding biophysical changes during disease progression  Week 9 Search Strategies in the Immune System Why Immune Cells Search - Targets are scarce in a relatively expansive system - Targets are beyond perceptual range - Direct contact with targets is necessary for function - Search purposes include: - Detecting damage - Detecting non-self entities - Detecting transformed cells Search Mechanisms - Cells employ different movement patterns: - Brownian movement - Lévy walk - Homing to affected tissues involves directed migration Cellular Machinery and Migration Motor Proteins and Cell Structure - Forces generated through: - Actin cortex - Microtubule network - Motor proteins - Cross-linkers - Cell cortex drives shape changes during: - Cell migration - Cell division - Apical constriction Migration Modes 1. Mesenchymal Migration: - Involves ECM interaction - Integrin-dependent - Uses RAC and CDC42 signaling 2. Amoeboid/Blebbing Migration: - RHOA-mediated contractility - Hydrostatic pressure-driven - Less dependent on adhesion Cytoskeletal Components - Actin structures:  - G-actin to F-actin conversion - Arp2/3-mediated nucleation for lamellipodia - Formin-mediated nucleation for filopodia - Membrane dynamics: - Bleb formation through hydrostatic pressure - Actomyosin contraction Cellular Communication During Immune Response Movement Responses to External Cues - Durokinesis/Durotaxis: Response to substrate rigidity - Haptokinesis/Haptotaxis: Response to immobilized ligands - Chemokinesis/Chemotaxis: Response to diffusing factors Swarming Behavior - Sequential process: 1. Initial cells locate infection/injury site 2. First responders secrete diffusing factors 3. Additional cells are recruited 4. Some neutrophils migrate to lymphoid organs - Regulation through GRK2-mediated GPCR desensitization Target Engagement and Killing Cytotoxic T Lymphocyte (CTL) Function - Direct engagement with target cells - Controlled perforation of target membrane - Delivery of apoptosis-inducing proteases - Specific targeting mechanisms: - Force application on target membrane - CTL lipid order - Uni-directional perforation Target Cell Interaction - Precise delivery of cytotoxic granules - Coordinated actin dynamics during engagement - Ensures specific targeting without collateral damage This comprehensive overview demonstrates how immune cells employ sophisticated mechanisms for searching, migrating, and engaging targets, highlighting the complex interplay between cellular mechanics and immune function.  Week 10 Fundamentals of Ion Channel Signaling - Ion channels are membrane proteins forming pores that allow specific ions to pass through - Channels are typically closed at rest and open probabilistically when stimulated - Ion flow is passive, driven by electrochemical gradients across the membrane - The membrane potential is maintained by: - Lipid membranes being impermeable to ions - Transporters establishing electrochemical gradients - Inside of cell being slightly more negative than outside (-90 to -40 mV) Types of Ion Channels and Gating Mechanisms - Channels can be gated by various stimuli: - Voltage - Ligands - Temperature - pH - Ions - Mechanical forces - Multiple stimuli (polymodal) Mechanosensitive (MS) Ion Channels Activation Mechanisms - Force from lipids: - Membrane stretch, compression, and curvature - Changes in tension - Force at lipid-protein interface - Force from tethers: - Protein connections to cytoskeleton or extracellular matrix - Displacement of tethers gates the channel PIEZO1 as a Model MS Channel - Identified in 2010 - Largest known ion channel with broad expression - Structure: - Homo-trimer - Forms tri-skelion shape - Contains mechanosensing domains - Can bend membranes - Characteristics: - Non-selective cation channel - Allows Ca++ and Na+ influx, K+ efflux - Rapid activation (