Stem Cells, Cloning, and Signaling Lec 6 Part 2 and Lec 7

Overview

Lecture covers tissue renewal, stem cells, cloning history, and cell signaling concepts.
Focus on key experiments (Till & McCulloch), stem cell types, reprogramming (Yamanaka factors), somatic cell nuclear transfer, and general principles of cell signaling.
Prepare by mastering concepts; specific signaling examples discussed in next lecture.

Till & McCulloch identified adult hematopoietic stem cells using mice.
Bone marrow transplants are an established stem cell therapy (hematopoietic replacement).
Embryonic stem cells (inner cell mass) are pluripotent: can form any adult cell type but not extra‑embryonic tissues (not totipotent).
Trophectoderm cells form extra‑embryonic tissues (placenta, umbilical cord).
Embryonic stem cells can be cultured indefinitely and induced to differentiate with morphogens (e.g., retinoic acid → neurons).
Historical problems with embryonic stem cell therapies: ethical concerns, immune rejection, and tumor risk from undifferentiated cells.

Somatic Cell Nuclear Transfer (SCNT): remove nucleus from unfertilized egg, insert nucleus from somatic cell → reconstructed zygote → embryo → embryonic stem cells or implantation for reproductive cloning.
SCNT produces cells/genome identical to donor nucleus (used for cloning animals).
Dolly the sheep (1996) = first cloned mammal via SCNT (mammary cell nucleus, surrogate female).
- Anecdotes: naming choice, all‑female steps, Dolly preserved for display.
SCNT is older technology; Nobel recognition dates back to earlier discoveries.

Shinya Yamanaka discovered reprogramming differentiated cells to pluripotency by introducing specific transcription factors.
Key idea: same genome; cellular identity depends on gene expression (transcription factor complement).
Yamanaka (OSKM) factors: Oct4, Sox2, Klf4, c-Myc (often memorized as OSKM or Yamanaka factors).
Introducing these transcription factors into adult cells (e.g., skin cells) reprograms them into induced pluripotent stem cells (iPSCs).
iPSCs sidestep ethical and immune issues: use patient cells, avoid fertilized embryos.
OSKM triggers cascade: chromatin remodeling, histone/DNA modifications, altered RNA/protein expression.
Plants: most plant cells are totipotent and can regenerate whole plants from somatic cells.

Extracellular Signaling Molecule: any molecule transmitted between cells (ions, proteins, hormones, gases).
Ligand: signaling molecule that binds a receptor.
Receptor: protein that binds ligand; can be cell surface (transmembrane) or intracellular.
Intracellular Signaling Molecule: transmits signals inside the cell; nonprotein ones are secondary messengers.
Secondary Messenger: small nonprotein intracellular signaling molecules (e.g., cAMP, Ca2+, DAG, IP3).
Effector: molecule that changes cell behavior (metabolism, shape, gene expression).
Input/Output: start (signal) and end (response) of a pathway.
Upstream/Downstream: relative order in a pathway.
iPSC: induced pluripotent stem cell (reprogrammed adult cell).

Short‑range signaling:
- Contact‑dependent: membrane‑bound ligand interacts with adjacent cell receptor (e.g., Delta‑Notch).
- Paracrine: local diffusible signals affect nearby cells.
- Autocrine: cell secretes signals that affect itself.
- Movement limited by uptake, degradation, diffusion limits, and receptor expression.
Long‑range signaling:
- Synaptic: neuron sends signal via long axon; synapse distance very short but transmission spans long distance overall.
- Endocrine: hormones secreted into bloodstream for body‑wide distribution.

Cell‑surface receptors bind hydrophilic ligands (cannot cross membrane).
Intracellular receptors bind small hydrophobic ligands (can cross membrane; often carried by carrier proteins in blood).
Target cell response requires expression of the matching receptor.

Pathways must be reversible and regulatable (on/off control).
Common molecular mechanisms:
- Phosphorylation (kinases add phosphate, usually activating; phosphatases remove phosphate, usually deactivating).
- GTP/GDP binding cycles (GEFs promote GDP→GTP activation; GAPs promote GTP hydrolysis to inactivate).
- Ubiquitination (E1/E2/E3 ligases attach ubiquitin; mono‑ or multi‑ubiquitination affects activity/localization; polyubiquitination targets proteasomal degradation).
- Secondary messenger production amplifies signals (e.g., cAMP, Ca2+, DAG, IP3).
Examples: kinase inhibitors reduce signaling output; GAP activators inhibit signaling by promoting GTP hydrolysis.

Signal transmission speed varies by mode (synaptic very fast; endocrine slower due to circulation).
Response speed depends on mechanism:
- Rapid: modification of existing proteins (e.g., phosphorylation), membrane potential changes.
- Slow: transcription and translation (require nuclear import, transcription, RNA processing, export, translation).
Some signals elicit both fast and slow responses.

Inhibitory relationships are independent; interpret sequential inhibition carefully.
- Example logic: Kinase inhibits inhibitor → inhibitor cannot inhibit transcription factor → transcription occurs.
- Avoid misreading as “inhibitor becomes active to inhibit” when upstream step actually prevents inhibition.
Practice reading network diagrams: treat each interaction independently and follow logical consequences.

Feedback modifies pathway dynamics and duration of response.
Positive feedback:
- Output or downstream component activates upstream element → amplifies/maintains signal.
- Can sustain output even after stimulus removal (cell memory).
Negative feedback:
- Downstream component inhibits upstream element → limits or terminates response.
- Delay in negative feedback produces different dynamics:
  - Long delay → oscillations (repeated rise and fall).
  - Short delay → transient peak followed by moderate steady state.
Graph interpretation: output (y‑axis) vs. time (x‑axis); identify presence and type of feedback by curve shape.

Problem: single signaling inputs could theoretically activate many downstream proteins; specificity prevents inappropriate activation.
Two main mechanisms for specificity:
1. Signaling Complexes
  - Preformed scaffold complexes: scaffold proteins bind and localize signaling proteins, limiting diffusion and ensuring ordered signaling.
  - Assembly on activated receptor: receptor phosphorylation creates docking sites that recruit specific signaling proteins only when active.
  - Phosphoinositide docking sites: receptor activity modifies membrane phosphoinositides, recruiting specific effectors to the membrane.
2. Coincidence Detectors
  - Target proteins require multiple inputs (e.g., two phosphorylation events) to become active.
  - Protein Y only active when both site A and site B are phosphorylated—ensures signal integration and conditional activation.
Protein interaction domains determine specificity:
- SH3 domains bind proline‑rich sequences.
- SH2 and PTB domains bind phosphorylated tyrosines, with specificity conferred by neighboring amino acids.
- PH domains bind specific phosphoinositides.
Domains are mixed and matched in proteins; domain composition predicts interaction partners and signaling roles.

Review these lecture concepts before next class; next lecture covers specific signaling examples.
Memorize OSKM (Oct4, Sox2, Klf4, c‑Myc) for induced pluripotency.
Practice interpreting signaling diagrams (inhibition chains, feedback loops, coincidence detectors).
Study secondary messengers (cAMP, Ca2+, DAG, IP3) and common regulatory mechanisms (kinases, phosphatases, GEFs/GAPs, ubiquitination).