BIO211 Exam practice: Blebbistatin & Myosin II

• Practice questions for a BIO211 Winter 2022 exam, in short answer format
• Questions test knowledge of molecular processes/pathways and their relation to cell behavior/development.
• Questions explain how disturbances affect mechanisms and cell dynamics in embryonic development.
• Precisely identifying the molecular mechanism is key including ordered steps and molecular details.
• Molecular details to include: protein names, phosphorylation, ubiquitylation, activation, and/or degradation.
• Predicting how cell behavior/embryonic development is affected accounts for some points
• Explaining why the cell behavior/embryonic development is affected accounts for some points

Blebbistatin and Myosin II

• Blebbistatin inhibits myosin II by preventing ATP hydrolysis by the myosin II heavy chain.
• ATP hydrolysis drives conformational change in myosin II, enabling the head to grab the next actin monomer.
• Inorganic phosphate release triggers the power stroke, leading to ADP loss and tight binding to actin.
• ATP binding relieves tight-binding, so blebbistatin prevents ATP hydrolysis, preventing myosin II from binding actin
• An 8-cell xenopus embryo segregates chromosomes via the mitotic spindle, but cannot complete cytokinesis if blebbistatin is introduced.
• Each of the 8 cells in the presence of blebbistatin will contain two nuclei, because myosin II can't generate the force to pinch cells apart.

Talin Mutation and Integrin-Actin Complexes

• Talin connects activated integrin receptors to actin filaments.
• Talin and kindlin are recruited to integrin receptors, then talin binds vinculin, connecting the complex to actin filaments.
• Force application pulls talin apart, revealing vinculin binding sites, creating a positive feedback loop.
• Talin mutation prevents conformation change in response to force, disrupting the positive feedback loop.
• Focal adhesion complexes stay small and can't generate the force to retract the trailing edge or allow the cell to protrude.
• Embryonic cell migration is completely stopped because of this.

Ciliobrevin D and Dynein

• Ciliobrevin D inhibits the dynein minus end microtubule motor.
• Dynein connects astral microtubules to the actin cytoskeleton during metaphase.
• Motor domains bind the plus ends of astral microtubules, while cargo binding domains connects to the actin cortex.
• Active dynein motors walk towards the minus end of the microtubule, pulling the centrosome and mitotic spindle towards the cell's edge.
• Balanced forces position the mitotic spindle towards the middle of the cell.
• Addition of ciliobrevin D will cause astral microtubules to no longer be pulled apart.
• Mitotic spindles are no longer positioned in the mid-point of the cell.
• Chromosomes may not be equally distributed between daughter cells during anaphase.

M-Cyclin Ubiquitylation Defect

• Cdc20 gets activated by proper positioning of mad2 and kinetochore attachment.
• APC is activated by Cdc20, but can't catalyze poly-ubiquitin chain addition to cyclin M without E1 and E2 ligases.
• Cyclin M isn't degraded, its expression persists, and cyclin-dependent kinase remains active because of this persistence.
• Overall, the embryo remains stuck at the metaphase-anaphase transition and can't segregate chromosomes, because of this.
• Loss of M cyclin enables the cell cycle to move through checkpoint, so its persistence halts this cellular movement.