In
cell biology
Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and ...
, single-cell variability occurs when individual
cells
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
in an otherwise similar population differ in shape, size, position in the
cell cycle, or molecular-level characteristics. Such differences can be detected using modern
single-cell analysis techniques. Investigation of variability within a population of cells contributes to understanding of
developmental and
pathological processes,
Single-cell analysis
A sample of cells may appear similar, but the cells can vary in their individual characteristics, such as shape and size,
mRNA expression levels,
genome, or individual counts of
metabolite
In biochemistry, a metabolite is an intermediate or end product of metabolism.
The term is usually used for small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, c ...
s. In the past, the only methods available for investigating such properties required a population of cells and provided an estimate of the characteristic of interest, averaged over the population, which could obscure important differences among the cells. Single-cell analysis allows scientists to study the properties of a single cell of interest with high accuracy, revealing individual differences among populations and offering new insights in molecular biology. These individual differences are important in fields such as developmental biology, where individual cells can take on different "
fates" - become specialized cells such as neurons or organ tissue - during the growth of an embryo; in
cancer research, where individual malignant cells can vary in their response to therapy; or in
infectious disease, where only a subset of cells in a population become infected by a
pathogen.
Population-level views of cells can offer a distorted view of the data by averaging out the properties of distinct subsets of cells. For example, if half the cells of a particular group are expressing high levels of a given gene, and the rest are expressing low levels, results from a population-wide analysis may appear as if all cells are expressing a medium level of the given gene. Thus, single-cell analysis allows researchers to study biological processes in finer detail and answer questions that could not have been addressed otherwise.
Types of variation
Variation in gene expression
Cells with identical genomes may vary in the
expression of their genes due to differences in their specialized function in the body, their timepoint in the
cell cycle, their environment, and also noise and
stochastic
Stochastic (, ) refers to the property of being well described by a random probability distribution. Although stochasticity and randomness are distinct in that the former refers to a modeling approach and the latter refers to phenomena themselv ...
factors. Thus, accurate measurement of gene expression in individual cells allows researchers to better understand these critical aspects of cellular biology. For example, early study of gene expression in individual cells in
fruit fly embryos allowed scientists to discover regularized patterns or gradients of specific gene transcription during different stages of growth, allowing for a more detailed understanding of development at the level of location and time. Another phenomenon in gene expression which could only be identified at the single cell level is oscillatory gene expression, in which a gene is expressed on and off periodically.
Single-cell gene expression is typically assayed using
RNA-seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
. After the cell has been isolated, the RNA-seq protocol typically consists of three steps: the RNA is
reverse transcribed into
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
, the cDNA is amplified to make more material available for the sequencer, and the cDNA is
sequenced.
Variation in DNA sequence
A population of single celled organisms like bacteria typically vary slightly in their
DNA sequence
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
due to
mutations acquired during reproduction. Within a single human, individual cells typically have identical genomes, though there are interesting exceptions, such as
B-cell
B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted o ...
s, which have variation in their DNA enabling them to generate different
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
to bind to the variety of pathogens that can attack the body. Measuring the differences and the rate of change in DNA content at the single-cell level can help scientists better understand how pathogens develop antibiotic resistance, why the immune system often cannot produce antibodies for rapidly mutating viruses like HIV, and other important phenomena.
Many technologies exist for sequencing genomes, but they are designed to use DNA from a population of cells rather than a single cell. The primary challenge for single-cell genome sequencing is to make multiple copies of (amplify) the DNA so that there is enough material available for the sequencer, a process called whole genome amplification (WGA). Typical methods for WGA consist of: (1) Multiple Displacement Amplification (MDA) in which multiple
primers anneal to the DNA,
polymerases copy the DNA, and knock off other polymerases, freeing strands that can be processed by the sequencer, (2)
PCR PCR or pcr may refer to:
Science
* Phosphocreatine, a phosphorylated creatine molecule
* Principal component regression, a statistical technique
Medicine
* Polymerase chain reaction
** COVID-19 testing, often performed using the polymerase chain r ...
-based methods, or (3) some combination of both.
Variation in
metabolomic
Metabolomics is the scientific study of chemical processes involving metabolites, the small molecule substrates, intermediates, and products of cell metabolism. Specifically, metabolomics is the "systematic study of the unique chemical fingerprin ...
properties
Cells vary in the
metabolites they contain, which are the intermediary compounds and end products of complex biochemical reactions that sustain the cell. Genetically identical cells in different conditions and environments can use different metabolic pathways to sustain themselves. By measuring the metabolites present, scientists can infer the metabolic pathways used, and infer useful information about the state of the cell. An example of this is found in the immune system, where
CD4+ cells can differentiate into Th17 or TReg cells (among other possibilities), both of which direct the immune system's response in different ways. Th17 cells stimulate a strong inflammatory response, whereas TReg cells stimulate the opposite effect. The former tend to rely much more on
glycolysis
Glycolysis is the metabolic pathway that converts glucose () into pyruvate (). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH ...
, due to their increased energy demands.
In order to profile the metabolic content of a cell, researchers must identify the cell of interest in the larger population, isolate it for analysis, quickly inhibit enzymes and halt the metabolic processes in the cell, and then use techniques such as
NMR,
mass-spec,
microfluidics, and other methods to analyze the contents of the cell.
Variation in proteome
Similar to variation in the metabolome, the
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
present in a cell and their abundances can vary from cell to cell in an otherwise similar population. While
transcription and
translation determine the amount and variety of proteins produced, these processes are imprecise, and cells have a number of mechanisms which can change or degrade proteins, allowing for variance in the proteome that may not be accounted for by variance in gene expression. Also, proteins have many other important features besides simply being present or absent, such as whether have undergone
posttranslational modifications such as
phosphorylation
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
, or are bound to molecules of interest. The variation in abundance and characteristics of proteins has implications for fields such as cancer research and cancer therapy, where a drug targeting a particular protein may vary in its impact due to variability in the proteome, or vary in efficacy due to the broader biological phenomenon of
tumor heterogeneity.
Cytometry, surface methods, and microfluidics technologies are the three classes of tools commonly used to profile the proteomes of individual cells.
Cytometry allows researchers to isolate cells of interest, and stain 15–30 proteins to measure their location and/or relative abundance.
[ Image cycling techniques have been developed to measure multi-target abundance and distribution in biopsy samples and tissues. In these methods, 3–4 targets are stained with fluorescently labeled antibodies, imaged, and then stripped of their fluorophores by a variety of means, including oxidation-based chemistries or more recently antibody-DNA conjugation methods, allowing additional targets to be stained in follow-on cycles; in some methods up to 60 individual targets have been visualized. For surface methods, researchers place a single cell on a surface coated with antibodies, which then bind to proteins secreted by the cell and allow them to be measured.][ Microfluidics methods for proteome analysis immobilize single cells on a microchip and use staining to measure the proteins of interest, or antibodies to bind to the proteins of interest.
]
Variation in cell size and morphology
Cells in an otherwise similar population can vary in their size and morphology due to differences in function, changes in metabolism, or simply being in different phases of the cell cycle or some other factor. For example, stem cells can divide asymmetrically, which means the two resultant daughter cells may have different fates (specialized functions), and can differ from each other in size or shape. Researchers who study development may be interested in tracking the physical characteristics of the individual progeny in a growing population in order to understand how stem cells differentiate into a complex tissue or organism over time.
Microscopy can be used to analyze cell size and morphology by obtaining high-quality images over time. These pictures will typically contain a population of cells, but algorithms can be applied to identify and track individual cells across multiple images. The algorithms must be able to process gigabytes of data to remove noise and summarize the relevant characteristics for the given research question.
Variation in cell cycle
Individual cells in a population will often be at different points in the cell cycle. Scientists who wish to understand characteristics of the cell at a particular point in the cycle would have difficulty using population-level estimates, since they would average measurements from cells at different stages. Also, understanding the cell cycle in individual diseased cells, like those in a tumor, is also important, since they will often have a very different cycle than healthy cells. Single-cell analysis of characteristics of the cell cycle allow scientists to understand these properties in greater detail.
Variability in cell cycle can be studied using several of the methods previously described. For example, cells in G2 will be quite large in size (as they are a just at the point where they are about to divide in two), and can be identified using protocols for cell size and shape. Cells in S phase
S phase (Synthesis Phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during ...
copy their genomes, and could be identified using protocols for staining DNA and measuring its content by flow cytometry or quantitative fluorescence microscopy, or by using probes for genes expressed highly at specific phases of the cell cycle.
References
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Cell biology