Week 8

• Bones and muscles are needed for body movement
• During movement, muscles contract but bones do not. However, the muscle applies force on the bones. So the question in mechanics is how bones withstand the force applied by muscles.
• When looking at the fiber properties, we realise muscles are relatively soft. There are three types of muscles: skeletal, cardiac and smooth. These muscles differ in structure and morphology despite containing the same genetic conditions.
• Bones are very rigid, even though they also are composed of cells. This is because of the different fibers involved in their formation.
• Generally, different cell types have different structure and properties.
• The difficulty faced in biomechanics is that all components are alive and hence, they can change structure, protein synthesis and properties, making them dynamic.
• Properties of cells depend on:

  1. What type of tissue
  2. What type of cell
  3. What type of structure they have

  Overall, the same set of genes are expressed into different set of proteins to determine mechanical properties.
• Macrophages can move and chase where the bacteria moves and engulf them in a process called phagocytosis.

  • for this to happen, the cell needs to grab onto the surface and move, applying force
  • They also need to change shape
• PROCESS OF PHAGOCYTOSIS

  Macrophage moves towards bacteria. Bacteria is engulfed. Bacteria is contained within a vesicle. Lysosome fuses with the vesicle and releases phagocytic enzymes. Bacteria are destroyed and digested. The vesicle in the end contains undigested remains of the bacteria.
• Cells like macrophages are about 50 micrometers. Bacterial cells, on the other hand, are about 0.5 micrometers.
• The long, thin pseudopod which have a thickness of 0.1 micrometers are the ones that pull the bacterial cells to the macrophages.
• After neurons have developed, it cannot divide. Hence, it cannot replace dead neurons and these cells must live very long. The neural circuit must be very robust to survive through these changes.
• This experiment uses different elastic substrates to show how the environment affects stem cell differentiation.

  
• From the experiment on stem cell differentiation, the following were observed:

  1. On softer substrates, stem cells developed long protusions like neurons.
  2. On very rigid substrates, they started to look like bone cells.

  This shows that cells can change their properties based on their environment.
• Endothelial cells are very responsive to mechanical forces generated by the circulatory system, including the shear from flow, stretch from the distension of vessels and transmural* pressure differences.

  *existing or occurring across the entire wall of an organ or blood vessel.
• The original shape of the endothelial cells is static shape. This occurs when there is no flow. Blood vessels are very responsive and are constantly change their radius and leakiness.
• Due to blood flow, there is constant shear force being applied on the endothelial cells. Due to fluid shear, they change their shape to align their long axis in the direction of the flow. This helps endothelial cells maintain blood flow and homeostasis.
• Normal tissue cells like fibroblasts wont be able to move too much. However, due to survival pressure, cancer cells have to expand and have to push forward. Hence, they are pushed to migrate. Highly malignant cancer cells have certain gene mutations that promote stronger binding to the ECM and have faster migration speed.
• Cancer metastasis is the process where by individual cancer cells detaches from the main tumour, enters the bloodstream, reattaches at at some new location, exits the blood vessel and starts growing in its new location. This process involves cell migration, which is mediated by mechanical processes such as adhesion and intracellular force generation.
• Adhesion may not only be important for allowing cancer cells to migrate but also for them to home in one particular location.
• During metastasis, cancer cells face mechanical challenge: they have to make a very small hole to enter the bloodstream. Normal cells are very rigid and hence, cannot squeeze through and will be blocked there. However, cancer cells are deformable but the less tough one will not survive the shear force caused by blood flow.
• When the ECM is softer, it applies lower nuclear stress allowing for cell migration during cancer metastasis.
• BASIC COMPONENTS OF MAMMALIAN CELLS

  
• Basic Components in cell mechanics:

  1. ECM: collagen fibers
  2. Membrane receptors: integrin, ion channel
  3. Cytoskeleton: actin filaments, Arp2/3, filamin
  4. Motor proteins: Myosin filaments
  5. Signalling proteins
  6. Nucleus: lamin proteins, chromatin, gene transcription regulation/transcription factors
• How cell migration occurs:

  1. G-actin polymerization
  2. F-actin filament assembly
  3. Arp 2/3 actin branching which pushes the cell forward
  4. Filamin cross-linking
  5. Cell protrudes forward along ECM fiber
  6. Integrin binds to ECM. Clutch protein anchor integrin to actin filaments.
  7. Integrin activates signalling protein. Signalling protein activates myosin.
  8. Myosin filament assemble. Myosin filament, actin filament, and alpha-actinin proteins assemble into large stress fibers.
  9. Stress fibers contract.

  After this, cell migration depends on the rigidity of the ECM.
• If it is a soft ECM, high ECM deformation results in low force exposure at focal adhesions. In rigid ECM, low ECM deformation results in high force exposure at focal adhesions.
• CELL MIGRATION (CONTINUED 1)

  In response to the force, focal adhesion proteins unfold and expose hidden binding. Bound proteins activate signalling proteins. Stretch-activated channels allow ion flow in response to force through auxiliary proteins (tether model) or via membrane pull (membrane tension model). Ion influx leads to cascade of signalling proteins that terminates at the nucleus.
• CELL MIGRATION (CONTINUED 2)

  Actomyosin contraction pulls directly on the nucleus via nuclear membrane protein. Lamin A aids in force propagation and interacts with chromatin. Transcription factors associated with nuclear membrane complex binds to chromatin. Transcription begins on the exposed gene. mRNA is transcribed from DNA. mRNA is exported from the nucleus and translated into new proteins at ribosomes. Newly expressed proteins result in new signalling cascades. Cell behaviour changes as a direct result of mechanotransduction.
• Extracellular Matrix Remodelling

  Cardiac tissue mainly consists of cells and strong fibers called collagen fibers, which are randomly distributed in the cell. When this tissue is likely loaded for prolonged periods of time, cells and collagen fibers align in the direction of the applied force. During this process, the cells can rotate and align parts of the fibrous matrix by pulling at the fibers. This process is called reorientation.
• Extracellular Matrix Remodelling (Continued 1)

  At the same time, the cell produces new collagen molecules that are transported outside the cell where they aggregate side by side into fibers and into thicker collagen bundles. Existing collagen is enzymatically degraded by cells. Collagen fibers that are not aligned in the direction of the applied forces are more prone to this degradation.
• Extracellular Matrix Remodelling (Continued 2)

  Part of the degraded collagen may be reused to strengthen existing collagen fibers at other local locations. The net result is an increased formation of collagen fibers in the direction of the applied load. When the load direction is changed again, cells and collagen fibers will remodel again.
• Challenges in cell mechanics:

  1. proteins, cellular structures and their dynamic regulation
  2. nN to pN force manipulation and measurement
  3. Imaging and Quantitative Analysis
• 1. What types of forces the cells may experience in the following scenarios?

  A cancer cell migrate through a dense ECM: compressive forces

  An endothelial cell on the blood vessel: shear forces

  A macrophage grabbing a moving bacteria: tensile forces
• 2. Components involved in cell migration:

  ECM, integrin receptors on cell membrane, actin cytoskeleton and myosin filaments
• What is the sequence of events when a cell pulls on a rigid ECM?

  1.Membrane receptor and/or its binding proteins may change their conformation

  2. More binding sites open for binding of signalling molecules

  3. cytosolic signalling activated

  4. signalling molecular transport into nucleus

  5. genetic regulation activated in nucleus
• What happens to ECM fibers when they are stretched and undergo ECM remodelling?

  ECM degradation if fibers does not align with stretching direction and ECM enhancement if fibers align with stretching direction
• What are the challenges for studying mechanics at the cellular level?

  • too small to directly visualize the parts by eyes
  • mostly colourless to differentiate parts
  • mostly in the force range pN to nN, hard to measure and manipulate
  • often coupled with genetic and signalling regulation in cells, thus complex to dissect
• How do the following technologies benefit the mechanobiology research?

  Genomic editing: enabling the precise modulation of the basic components of cells

  Optical tweezers: enabling the measurement and manipulation of forces at pN and nN scale

  Super-resolved fluorescence microscopy: enabling imaging and quantitative analysis of cellular components