Cell Cycle and Morphogen Gradients Quiz

Cell Signaling Principles

• Cell phenotype is determined by gene expression into mRNA and protein. Different cell types have distinct gene expression profiles.
• Development involves growth and patterning, with patterning regulated by coordinated cell signaling between different cell types.
• Key cell processes involved in development include proliferation, specialization, interaction, and movement.
• The zygote (fertilized egg) differentiates into three germ layers (ectoderm, mesoderm, and endoderm) that form all other tissues.

Generating Cell Diversity

• Cells diversify while still in precursor stages, primarily from intrinsic cellular information.
• Cell division does not equally divide contents like mRNA and cytokines, leading to varying identities in daughter cells.

Cellular Interactions Model

• Cells physically interact and signals are passed externally to surrounding cells which influences cell behavior.
• Cells have the ability to develop in different ways based on the concentration of signalling molecules (gradients).

Direct Morphogen Gradient

• Localized production of an inducer forms a gradient from its source.

Indirect Morphogen Gradient

• Localized production of an inhibitor diffusing outward blocks the action of a uniformly distributed inducer. This creates a gradient.

Patterning by Sequential Induction

• Inductive interactions allow for the formation of many diverse cell types, forming intermediate zones by cell interactions with signals.

Cutting Tissue

• Removing tissue from its natural environment can cause differentiation, like extracting endoderm from zygotes generating endodermal and mesodermal cells.

Spinal Cord Patterning

• Spinal cord pattern is controlled by Shh/BMP signals coming from the dorsal (roof plate) and ventral (floor plate) sides, respectively. These gradients determine neuronal subtype differentiation.

Different Ways of Transmitting Signals

• Molecules or electric signals can pass through pores/receptors.
• Direct contact between two cells.
• Gap junctions allow communication between cells.

Receptor-Mediated Signaling

• Extracellular molecules interact with receptor proteins, initiating intracellular signaling cascades. Important proteins include enzymes, regulatory proteins, or cytoskeletal proteins. This leads to alterations in metabolism, gene expression, and/or cell shape/movement.
• Examples include MAPK pathways where a stimulus triggers a series of phosphorylations that lead to cellular responses..

Wnt Pathway - β-catenin Pathway

• Wnt signaling is an on/off system that determines body axis and specific developmental processes.
• The presence or absence of the Wnt ligand affects the level of β-catenin, a protein that influences gene expression.

TGF-β Pathway

• TGF-β signaling involves two receptor types that only bind when the ligand is present.
• The phosphorylation cascade, initiated by Smad2/3 and Co-Smad2, regulates transcription of target genes.
• Important for left-right body asymmetry and skeleton formation.

Hedgehog Pathway

• Hedgehog proteins (Shh, Ihh, Dhh) affect target genes via intracellular signaling and regulate development.
• In the presence of hedgehog, the protein Ci will not be blocked. It then activates target genes.

Receptor Tyrosine Kinase Pathway

• Receptors have intracellular tyrosine kinase components, initiating phosphorylation cascades.
• Pathways involve proteins like PDGF, FGF and Eph receptors.
• Related to cancer development when certain proteins are mutated.

Notch/Delta Pathway

• Cell-cell contact is important for differentiation, where one cell "inhibits" another from doing a specific thing.
• Signaling through Notch and Delta proteins determines cell fate.
• Involved in development of the nervous system, retina, and blood cells.

Cell Proliferation Analysis

• Cell cycle is regulated by cyclin-dependent kinases (CDKs) and cyclins.
• The content of the cell changes during cell division, with organelles being equally distributed between daughter cells.
• Telomeres, which shorten with each division, can induce a crisis that some cells can overcome.
• Telomerase can re-synthesize telomeres, allowing cells to replicate indefinitely.
• Quiescence cells are those that have stopped dividing but can re-enter the cell cycle, while differentiated cells typically do not divide.

Physiological Factors for Stopping Proliferation

• Metabolic stimuli/starvation.
• Confluency.
• Chemical compounds (mitomycin C, camptothecin).
• Mechanical stimuli.
• Cellular ageing.
• Transformation/tumorigenesis.

Cell Growth Measurements

• Cell proliferation assays measure cell growth, including counting cells with Bürker chambers.
• Population doubling time (PDT) measures the time required for cell number to double.

Cell Cycle Analysis

• Propidium iodide (PI) is a red fluorescent DNA stain used to analyze different phases of the cell cycle.
• Flow cytometry can quantify DNA content per cell and determine the percentage of cells in each phase of the cell cycle including G0/G1, S, G2/M phases..
• Cell cycle analysis can be used to determine the proliferation capacity of cells undergoing treatments.

Cell Proliferation Marker Analysis

• PCNA, Ki67, and histone H3 are markers expressed in different phases of the cell cycle (G1, G2, S, and M).
• PCNA is present in cell cycle S and G1 phase
• pHH3 marks the percentage of cells in the M phase.
• Ki67 marks the cycling cells in G1, S, G2 and M phase.

Dye Dilution Assays

• CFSE (carboxyfluorescein diacetate succinimidyl ester) is a dye that can be used to measure cellular proliferation.
• The dilution of CFSE over cell division allows quantification of the proliferation rates.

Protein Marker Detection Techniques

• Protein markers identify cell types and functions.
• Techniques for detection include Western blotting and dot blots for qualitative analyses.
• Immunostaining allows detection of where proteins are expressed within samples and is good for qualitative analyses.

Proteomics Approach

• Proteomics systematically characterizes proteins in a sample.
• Protein identification is done by extracting proteins, digesting, separating into peptides, and analyzing them using mass spectrometry.
• Comparing results with databases can identify peptides and proteins and determine how these are being composed.

Protein Immunodetection Approach

• Immunostaining is used to detect the presence and spatial distribution of a protein target.
• ELISA is used for qualitative/quantitative detection of specific proteins in samples via monoclonal antibodies that recognise the target.
• Microarray is useful for large-scale screening of protein targets since it immobilizes antibodies onto a slide to detect various proteins in a sample simultaneously.

Single-Cell Techniques for Protein Detection

• Flow cytometry detects the physical and/or fluorescent properties of individual cells in a fluid sample to give an image. Specific markers or antibodies can be used to target cells that express specific properties and/or proteins.
• Fluorescent antibodies which can be excited at determined wavelengths are used to label cells.
• The antibodies can be directly attached to a fluorophore or indirectly through an antibody-based indirect coloration.
• Intracellular markers require permeabilizing the cell membrane to allow access to the target.
• Surface markers are in the cell's outer surface and are fully accessible and detected directly by the antibody

Histogram modality (mono-parametric analysis)

• Analysing the data using one parameter at a time (one fluorescence channel) showing cell populations, mix population, and subgroups based on their fluorescence intensity.

Dot plot (multi-parametric analysis)

• Each dot represents a cell and uses multiple fluorescence channels to visualize the cell populations that express multiple markers simultaneously. It is better when there are more than one parameter.

Cell Sorting: FACS (fluorescence-activated cell sorting)

• Separates cell types based on size, granularity, and/or fluorescence intensity.
• Cells with a particular combination of characteristics can be separated, collected, and used in further analyses.

Mass Cytometry (CyTOF)

• Uses antibodies labeled with metal isotopes, enabling simultaneous analysis of many markers.
• Measures the time each isotope takes to travel through an electric field. This method is more sensitive to the multiplexing of the different labelled markers.

Imaging Flow Cytometry

• Combines cell imaging with flow cytometric analysis.
• Provides information about cell morphology and/or labelling localization/intensity

Magnetic Activated Cell Sorting (MACS)

• Non-cytometric method that uses magnetic beads and a column to sort cells based on markers.
• It doesn't destroy the cells as it is a gentler method.

Cell Health Assays

• Used to assess the proportion of live cells within a population.
• Trypan blue exclusion method.
• Nuclear ID dual dye method.
• Esterase activity and membrane integrity.
• ATP assay.
• MTT assay.

Cell Viability Assays - Apoptosis Assays

• Measures programmed cell death (apoptosis) through different methods:
• Plasma membrane integrity assay (Phosphatidylserine (PS) and Annexin V labeling).
• TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling).
• Apoptotic markers (caspases).

Other Cell Viability Assays-Cell Senescence

• Cell senescence is an irreversible loss of replicative potential often due to DNA damage.
• Cell morphology can be used to detect senescence.
• Senescence-associated B-galactosidase (SA-B-gal) assay
• Raman microscopy can identify the chemical composition of cells and distinguish living and dead or different cell stages.

Gene Expression Analysis - RNA Markers

• Northern blotting detects RNA markers
• RT-PCR (reverse transcription polymerase chain reaction) amplifies specific mRNA sequences.
• Quantitative RT-PCR (qPCR) quantifies the amount of mRNA.
• TaqMan probe is used for qPCR.
• Microarrays enable high-throughput analysis of thousands of genes at once.
• RNA-sequencing (RNA-seq): a high-throughput technique that determines transcripts from the entire RNA population of the cells.

RNA Markers - Spatial/Localized Detection

• Laser capture microdissection targets genes in specific areas of the sample.
• In situ hybridization (ISH): determines if mRNA is present or not and where in the sample it is present.
• Multiplexed target Survey: a combination of different techniques.

Test your knowledge on the cell cycle phases, specifically the S phase, and the concepts of morphogen gradients. This quiz covers topics such as DNA content analysis, signaling methods, and tissue patterning. Challenge yourself with questions related to cellular interactions and the role of inducers in morphogen gradients.