Cell Cycle and Morphogen Gradients Quiz

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What state of DNA content is observed during the S phase of the cell cycle?

Each cell has a single copy of DNA.

Each cell has variable amounts of DNA.

Each cell has no DNA present.

Each cell has two copies of DNA. (correct)

Which method can be used to analyze the DNA content and cell cycle stage?

Flow cytometry (correct)

PCR amplification

Gel electrophoresis

Western blotting

What marker is used in the original method to label DNA during the S phase?

Fluorescein isothiocyanate

Adenosine triphosphate

5-bromo-2'-deoxyuridine

3H-thymidine (correct)

What happens to the amount of labelling in DNA with each cell division when H3-thymidine is present?

The amount of labelling doubles. Signup and view all the answers

In which cell cycle phase is there a lower peak observed in flow cytometry compared to G0/G1?

G2/M phase Signup and view all the answers

What characterizes a direct morphogen gradient?

Inducer diffuses from a localized source. Signup and view all the answers

How does an indirect morphogen gradient function?

It involves a uniformly distributed inducer being blocked. Signup and view all the answers

What is the result of cutting tissue and removing it from neighboring cells?

It leads to specialized cell types based on location. Signup and view all the answers

What defines spinal cord patterning in terms of signaling?

Signals from the dorsal side influence differentiation. Signup and view all the answers

How is diversity generated in patterning by sequential induction?

From intermediate zones formed by neighboring cell interactions. Signup and view all the answers

What methods are included in transmitting signals between cells?

Diffusion via pores, direct contact, and gap junctions. Signup and view all the answers

Which of the following best describes the outcome of direct contact between different cell types?

It generates various unique cell types through interaction. Signup and view all the answers

What effect does a localized inducer have in a morphogen gradient?

It creates a gradient of activity influenced by an inhibitor. Signup and view all the answers

What is the purpose of using a housekeeping protein in protein quantification?

To normalize the signal across samples Signup and view all the answers

Which of the following steps is NOT part of the dot blot method?

Protein separation by gel electrophoresis Signup and view all the answers

In a Sandwich ELISA, how is the target protein detected?

Using a capture antibody and a detection antibody with a signaling tag Signup and view all the answers

What is the main advantage of using protein microarrays?

Analyzing multiple proteins simultaneously Signup and view all the answers

What does immunostaining primarily provide information about?

The presence and spatial distribution of a target protein Signup and view all the answers

Which of the following best describes a bulk detection method?

Provides qualitative assessment of protein expression Signup and view all the answers

How does the dot blot method differ from traditional Western blotting?

Dot blot applies samples directly onto a membrane Signup and view all the answers

What is typically immobilized on a slide in protein microarrays?

Specific antibodies for various proteins Signup and view all the answers

What is the primary purpose of sample fixation in immunostaining?

To preserve the structure and components of the sample Signup and view all the answers

Why is the indirect detection method in immunostaining preferred over the direct method?

It amplifies the signal more effectively Signup and view all the answers

What role does the secondary antibody play in the immunostaining process?

It recognizes the primary antibody and carries a detection tag Signup and view all the answers

In the context of immunostaining, what is the purpose of the positive control?

To ensure the detection method is functioning correctly Signup and view all the answers

What might be a consequence of not including a negative control in an immunostaining experiment?

False positives might go undetected Signup and view all the answers

What effect does using multiple secondary antibodies have in the immunostaining process?

It enhances signal amplification and brightness Signup and view all the answers

Which detection methods are utilized in immunostaining procedures?

Both direct and indirect methods with various labels Signup and view all the answers

What is a significant advantage of using fluorochrome conjugated antibodies in immunostaining?

They provide highly specific signals with less background noise Signup and view all the answers

What is a characteristic feature of cells that have entered senescence?

Irregular borders and being very spread out Signup and view all the answers

Which enzyme is used as a marker to identify senescent cells?

Senescence-associated ß-galactosidase Signup and view all the answers

Why is the new fluorescent substrate superior to the substrate X-Gal for identifying senescent cells?

It is suitable for real-time imaging of live cells Signup and view all the answers

What technique is used to create a chemical fingerprint of a sample at the single-cell level?

Confocal Raman Microscopy Signup and view all the answers

What is the main principle behind Raman spectroscopy?

Scattering of laser light with frequency shift Signup and view all the answers

What happens to cells that reach the end of their replicative capacity?

They enter senescence and stop replicating Signup and view all the answers

Which of the following techniques is NOT suitable for real-time imaging of senescent cells?

Microscopic examination of cellular morphology Signup and view all the answers

How can luminescence be quantitatively measured?

By luminometry Signup and view all the answers

What does the presence of cells on the other side of the Matrigel indicate?

Cells can reorganize their extracellular matrix. Signup and view all the answers

Which type of microscope is primarily used for large samples like organs or embryos?

Stereomicroscope Signup and view all the answers

What is the magnification range of a stereomicroscope?

20-100X Signup and view all the answers

Which microscope allows the observation of cell cultures with minimal distance to the sample?

Inverted compound microscope Signup and view all the answers

What advantage does phase contrast microscopy provide?

It improves the visibility of edges by amplifying contrast. Signup and view all the answers

What is the function of a semi-permeable membrane in the described assay?

It allows the evaluation of cell migration capability. Signup and view all the answers

Why is the compound microscope not recommended for live cell imaging?

It has a risk of damaging the live samples. Signup and view all the answers

What does the term 'resolution' refer to in microscopy?

The ability to distinguish between two separate points. Signup and view all the answers

Study Notes

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.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

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.