At the recent Widnes SciBar presentation, Dr. Duncan Casey from Liverpool John Moores University introduced the audience to developments in single cell manipulation and interrogation using experimental and theoretical ideas drawn from Physics, Chemistry and Biology. The work forms part of an ongoing collaboration with groups at Imperial College London and Glasgow University. Dr. Casey moved from Imperial to Liverpool, where he now holds an academic position in the Department of Civil Engineering.
The recent moves away from the classical approaches used in Biochemistry, describing phenomena on the basis of whole cell populations (sometimes tissues, often cultured cells, such as HeLa cells), towards a single cell approach, is similar to experimental efforts to investigate single protein molecules, instead of populations. However, both objectives require the development of methods and instrumentation in which the scale of the cell (or the protein) can be "matched" by the physical phenomenon. In Dr. Casey's presentation we were introduced to the use of silica beads coated, in one example, in a lipid formulation, in order to "pluck" membrane associated proteins from inside a single cell for analysis. In one application, a single "bad apple" in a population of cells, may represent the early stage of a tumour: this is an ideal opportunity to understand and "attack" the differences, perhaps before it's too late. Clearly, single cell "surgery" would represent an enormous step forward in medicine, however, before I continue, let me try and explain some of the background, in particular, the metrics, the physics and the biochemistry, for those who couldn't make the talk.
Single cells in humans are on average around 15 microns in diameter, i.e. 15 millionths of a meter. Or, if you prefer, a pinhead is about 1500 microns, i.e. 100 cells across. Depending on the function of the cell, it may be spherical or squashed nearly flat and in some cases of specialised cells, such as neurons they can be much larger. The HeLa cells used in Dr. Casey's work were derived over 60 years ago from a cervical cancer and have been continually cultured ever since! As Dr. Casey pointed out, the genome (simply realised at one level by the "karyotype" of the cell), has become rather abnormal with time. Nevertheless, HeLa cells are viewed by most as a work-horse for cell biologist, since despite their unorthodox chromosomal composition, they behave like an "average" human cell in many experimental situations. For reference, the general structure and organisation of a mammalian cell is shown below. For the purposes of this post, the key feature to note is the plasma membrane.
The experiments described by Dr. Casey involved the use of HeLa cells spread in a single layer on a suitable surface (typically plastic, glass or similar), imagine a microscope slide mounted under a microscope lens: the viewing lens is typically above the slide and illumination is provided from below. The next thing to consider is the chemistry of the coated beads that are going to provide the link between the laser and the cellular surgery.
Silicon beads have been widely used in chemistry and biochemistry for over 30 years. My own lab has used them extensively as the support for the separation of Biological Molecules. In this work, the beads are coated with a hydrophobic, "artificial membrane", which provides the required surface chemistry for attachment of the coated beads to the plasma membrane: such compounds include the well used molecule Triton X100 (below). The beads are approximately 1 micron in diameter, therefore around 5-10 beads can be accommodated along the equator of an immobilised cell (assuming the underside is unavailable). I am sure you can work out the maximum number of 1 micron diameter spheres needed to coat a sphere of diameter 15 microns. The plasma membrane can be described in two ways. First it is formed by the association of two phospholipid
molecules organised tail-to-tail: these individual molecules form a lipid bilayer. This "vessicle", is then peppered with protein molecules, in a cell-specific manner. Some of these proteins span the membrane, some are mostly on the outside, some on the inside and there are others that form transient interactions with the inside of the bilayer. The details of the so called plasma membrane of a typical mammalian cell are shown below.
In the experiment described in some detail by Dr. Casey, an artificially engineered Green Fluorescent Protein (GFP), fused to a protein that is known to associate with the inner surface of the membrane, was targeted for a proof of principal "grab and go" experiment (the Peripheral protein in the bottom left of the above figure is similar to the targeted GFP). Dr. Casey likened this to spear fishing: fish that swim close to the surface of the water are easily speared, without the need for a rod and line. The idea is that the coated bead fuses transiently with the lipid bilayer and sometimes, as it leaves the cell surface, it will bring with it a GFP molecule that happens to be in the vicinity, by exchanging the membrane binding moiety of the engineered GFP, for the Triton coated bead. As its name suggests, GFP has the convenient property of naturally "glowing in the dark", making the whole experiment relatively easy to monitor with common cell imaging equipment. Dr. Casey showed a nice video of the experiment in situ.
So we have covered the cell biology, we have an appreciation of the chemistry and the scale of operations, what of the Physics? As Dr. Casey pointed out in his title, optics are an important aspect of his work. If the micro-beads (and it should be noted the silica beads are not nano-particles), can be maneuvered into position on the cell surface, then a level of control can be introduced, which converts this technology from a dynamic, stochastic membrane fusion process, to one that can be claimed as precision cell surgery. The phenomenon that Dr. Casey described to manipulate the microbeads relies on the use of lasers: importantly, non-invasive lasers with light of wavelengths around 1000nm. This is typically the wavelength used for TV remotes, and is clearly harmless (apart from allegedly promoting obesity and a general level of human inactivity!). A laser (LASER, for definition: light amplification by stimulated emission of radiation; possibly the best acronym ever!) as you will all be aware, facilitates the focusing of a coherent beam of light (or electromagnetic radiation), and, by tuning the wavelength to that of the particle in its path (partly by the choice of oscillating material, Ytterbium in this case), a force is imparted upon the particle in such a way that it can be harnessed to to manipulate the particle. [I like this short animation that explains the basis of lasers which you can view here.] In this case an infrared laser emitting em radiation of 1017nm is used to push the bead(s) around. If you have ever played magnetic football, I think you will get the idea!
The instrumentation is a little more sophisticated than my description above, moreover, it is possible to apply relatively simple "code" to generate quite elaborate optical patterns including holograms. The schematic design of the instrument is shown below, and was discussed in some detail by Dr. Casey.
The final part of the talk focused on the use of a methodology in conjunction with a team at Glasgow, of controlled introduction of genetic material, DNA and possibly messenger RNA, the protein code itself, in order to instruct cells to behave in a certain way. (See later for the discussion). All cells possess chromosomes: bacteria often possess a single, circular chromosome, while human cells, for example, possess 23 pairs of (X-shaped) chromosomes and plant cells often possess many more. Since the development of molecular cloning in the 1970s, one of the essential methods has been the introduction of defined nucleic acid molecules into cells. In bacteria this is achieved mostly by harnessing a natural phenomenon called transformation in which "mini chromosomes" called plasmids are used to carry foreign genes into the host cell. This process occurs naturally as a means of genetic exchange between bacteria of the same or different species, and contributes to antibiotic resistance. In mammalian cells, the introduction of cloned genes is achieved in a similar manner by a process called transfection. In both cases, an excess of DNA molecules, typically small circular plasmids encoding around half a dozen gene functions are introduced into the cell either by the addition of something of a black box cocktail of chemicals, or in some situations by the application of a high (localised) voltage. In both cases the selection of those cells that have both survived the procedure, and taken up the DNA, forms an important part of any analysis of the functional consequences.
Dr. Casey laid out a vision for the controlled introduction of nucleic acids into cells. Why is this so important? In 1928, Fred Griffiths, working for the Ministry of Health, established that bacterial cells (Pneumococci) could be "transformed" from one strain type to another, by an ill-defined biological extract. 16 years later at the Rockefeller Institute in New York, Avery, Macleod and McCarty demonstrated that the transforming factor was DNA. (A later, technically more robust experiment by Hershey and Chase, nailed this unequivocally, when they took advantage of te new availability of stable isotopes). The limited experimental control over transformation or transfection of cells, is frustrating. Any innovation in this area has the potential to radically influence the rate of progress in gene therapy. For example, it may be to change a cancer-like phenotype, to a benign one, or develop new strains of bacteria with useful properties. The figure above shows cells transfected with genes expressing green and red versions of GFP!
Dr. Casey explained the limitations of current electroporation technology, in which plasmid like DNA molecules are transferred largely by chance and brute force, through electrostatic "holes" punched into the plasma membrane. This technology and the complementary chemo-transfection methods have underpinned our understanding of genes and their expression in human cells for the last three decades. However, as interdisciplinary funding matures, more opportunities are emerging to bring greater levels of experimental control to these methods. In collaboration with Professor Cooper's group at Glasgow, Dr. Casey demonstrated how combining the laser technology developed for "cell-surgery" with the lab-on-a-chip work in Glasgow, was yielding good progress in reducing the risk to cell physiology associated with conventional electroporation, and offers the prospect of a more controlled approach to addressable delivery of macromolecules such as DNA for both research based genetic intervention and possibly therapeutic intervention in the future.
Not surprisingly perhaps, as is the tradition at the Widnes Sci Bar (an audience that any Scientist should never underestimate!), Dr. Casey fielded questions relating to the laser technology and importantly the physical arrangement of the instrumentation to help appreciate the practicalities and the scale of these emerging technologies. In addition it should be noted that there are considerable challenges in "handling" nucleic acids, by whatever means, in view
of their susceptibility to shear and enzymatic degradation. The new technologies coming out of the Venter Lab, for example, in which whole genomes are being "installed" in bacterial hosts, would benefit enormously from the developments discussed. Craig Venter is pictured with key team member, the Nobel Laureate, Ham Smith. Finally, as always, Bob Roach our enthusiastic MC for the evening invited Dr. Duncan to comment on his vision for the future prospects of "light saber" technology. The possibility of "fixing" single gene defects such as cystic fibrosis (CF) through the controlled introduction of the messenger RNA encoding the "normal" protein via coated silicon beads in an inhaler-like device, followed by a "safe" laser blast, was something he envisaged might be an outcome of this work. Certainly, CF has been a "favourite" target for gene therapy in view of the relatively simple phenotype of the CFTR gene [the protein regulates transmembrane chloride ion exchange and in the West, patients make the protein, in a mutated form, which leaves it stuck inside the cell, when it needs to be in the cell membrane, to fulfill its normal Biological role]. I am not so sure how well the lung epithelia will deal with a fine spray of micro-beads, but I assume the chemists will rise to that challenge should the therapeutic opportunity become compelling. And finally, I hope this post helps with the understanding of some of the background to Dr. Duncan's presentation, and provides a taster fort those who couldn't make it, and another vibrant evening at the Hillcrest was had by all!
I'll update the post with some Open Access supporting articles over the weekend! And....as always, these are my own views and comments, queries corrections are always welcome and never ignored!
HeLa cells: experimental human derived cell line
Silica beads: micro scale spherical beads used in separation science, that can be coated with defined chemistries
Genome: the full complement of genes required for faithful copying of a cell or organism
Karyotype: the complement and morphology of the set of chromosomes in a eukaryotic cell. The number and gross structure of the microscopically examined chromosomes often reveal abnormalities which may be linked to certain genetic diseases
Membrane: the physical boundary around a cell. In animal cells it comprises hospholipids in a bilayer, interspersed with protein molecules
Green fluorescent protein (GFP): A naturally occurring protein isolated from jelly fish, that fluoresces bright green in natural light. It is used as a reporter for many cell functions
Transformation: the natural or experimental process of changing the characteristics of a bacterial cell by the addition of genetic material, usually DNA
Transfection: the equivalent of transformation in eukaryotic cells
Ribonucleic acid (RNA): the nucleic acid that contains the code for (usually) single proteins in all cells. It comes in a number of forms which are currently the focus of a great deal of research interest
The recent moves away from the classical approaches used in Biochemistry, describing phenomena on the basis of whole cell populations (sometimes tissues, often cultured cells, such as HeLa cells), towards a single cell approach, is similar to experimental efforts to investigate single protein molecules, instead of populations. However, both objectives require the development of methods and instrumentation in which the scale of the cell (or the protein) can be "matched" by the physical phenomenon. In Dr. Casey's presentation we were introduced to the use of silica beads coated, in one example, in a lipid formulation, in order to "pluck" membrane associated proteins from inside a single cell for analysis. In one application, a single "bad apple" in a population of cells, may represent the early stage of a tumour: this is an ideal opportunity to understand and "attack" the differences, perhaps before it's too late. Clearly, single cell "surgery" would represent an enormous step forward in medicine, however, before I continue, let me try and explain some of the background, in particular, the metrics, the physics and the biochemistry, for those who couldn't make the talk.
Single cells in humans are on average around 15 microns in diameter, i.e. 15 millionths of a meter. Or, if you prefer, a pinhead is about 1500 microns, i.e. 100 cells across. Depending on the function of the cell, it may be spherical or squashed nearly flat and in some cases of specialised cells, such as neurons they can be much larger. The HeLa cells used in Dr. Casey's work were derived over 60 years ago from a cervical cancer and have been continually cultured ever since! As Dr. Casey pointed out, the genome (simply realised at one level by the "karyotype" of the cell), has become rather abnormal with time. Nevertheless, HeLa cells are viewed by most as a work-horse for cell biologist, since despite their unorthodox chromosomal composition, they behave like an "average" human cell in many experimental situations. For reference, the general structure and organisation of a mammalian cell is shown below. For the purposes of this post, the key feature to note is the plasma membrane.The experiments described by Dr. Casey involved the use of HeLa cells spread in a single layer on a suitable surface (typically plastic, glass or similar), imagine a microscope slide mounted under a microscope lens: the viewing lens is typically above the slide and illumination is provided from below. The next thing to consider is the chemistry of the coated beads that are going to provide the link between the laser and the cellular surgery.
molecules organised tail-to-tail: these individual molecules form a lipid bilayer. This "vessicle", is then peppered with protein molecules, in a cell-specific manner. Some of these proteins span the membrane, some are mostly on the outside, some on the inside and there are others that form transient interactions with the inside of the bilayer. The details of the so called plasma membrane of a typical mammalian cell are shown below.In the experiment described in some detail by Dr. Casey, an artificially engineered Green Fluorescent Protein (GFP), fused to a protein that is known to associate with the inner surface of the membrane, was targeted for a proof of principal "grab and go" experiment (the Peripheral protein in the bottom left of the above figure is similar to the targeted GFP). Dr. Casey likened this to spear fishing: fish that swim close to the surface of the water are easily speared, without the need for a rod and line. The idea is that the coated bead fuses transiently with the lipid bilayer and sometimes, as it leaves the cell surface, it will bring with it a GFP molecule that happens to be in the vicinity, by exchanging the membrane binding moiety of the engineered GFP, for the Triton coated bead. As its name suggests, GFP has the convenient property of naturally "glowing in the dark", making the whole experiment relatively easy to monitor with common cell imaging equipment. Dr. Casey showed a nice video of the experiment in situ.So we have covered the cell biology, we have an appreciation of the chemistry and the scale of operations, what of the Physics? As Dr. Casey pointed out in his title, optics are an important aspect of his work. If the micro-beads (and it should be noted the silica beads are not nano-particles), can be maneuvered into position on the cell surface, then a level of control can be introduced, which converts this technology from a dynamic, stochastic membrane fusion process, to one that can be claimed as precision cell surgery. The phenomenon that Dr. Casey described to manipulate the microbeads relies on the use of lasers: importantly, non-invasive lasers with light of wavelengths around 1000nm. This is typically the wavelength used for TV remotes, and is clearly harmless (apart from allegedly promoting obesity and a general level of human inactivity!). A laser (LASER, for definition: light amplification by stimulated emission of radiation; possibly the best acronym ever!) as you will all be aware, facilitates the focusing of a coherent beam of light (or electromagnetic radiation), and, by tuning the wavelength to that of the particle in its path (partly by the choice of oscillating material, Ytterbium in this case), a force is imparted upon the particle in such a way that it can be harnessed to to manipulate the particle. [I like this short animation that explains the basis of lasers which you can view here.] In this case an infrared laser emitting em radiation of 1017nm is used to push the bead(s) around. If you have ever played magnetic football, I think you will get the idea!
The instrumentation is a little more sophisticated than my description above, moreover, it is possible to apply relatively simple "code" to generate quite elaborate optical patterns including holograms. The schematic design of the instrument is shown below, and was discussed in some detail by Dr. Casey.
The final part of the talk focused on the use of a methodology in conjunction with a team at Glasgow, of controlled introduction of genetic material, DNA and possibly messenger RNA, the protein code itself, in order to instruct cells to behave in a certain way. (See later for the discussion). All cells possess chromosomes: bacteria often possess a single, circular chromosome, while human cells, for example, possess 23 pairs of (X-shaped) chromosomes and plant cells often possess many more. Since the development of molecular cloning in the 1970s, one of the essential methods has been the introduction of defined nucleic acid molecules into cells. In bacteria this is achieved mostly by harnessing a natural phenomenon called transformation in which "mini chromosomes" called plasmids are used to carry foreign genes into the host cell. This process occurs naturally as a means of genetic exchange between bacteria of the same or different species, and contributes to antibiotic resistance. In mammalian cells, the introduction of cloned genes is achieved in a similar manner by a process called transfection. In both cases, an excess of DNA molecules, typically small circular plasmids encoding around half a dozen gene functions are introduced into the cell either by the addition of something of a black box cocktail of chemicals, or in some situations by the application of a high (localised) voltage. In both cases the selection of those cells that have both survived the procedure, and taken up the DNA, forms an important part of any analysis of the functional consequences.
Dr. Casey laid out a vision for the controlled introduction of nucleic acids into cells. Why is this so important? In 1928, Fred Griffiths, working for the Ministry of Health, established that bacterial cells (Pneumococci) could be "transformed" from one strain type to another, by an ill-defined biological extract. 16 years later at the Rockefeller Institute in New York, Avery, Macleod and McCarty demonstrated that the transforming factor was DNA. (A later, technically more robust experiment by Hershey and Chase, nailed this unequivocally, when they took advantage of te new availability of stable isotopes). The limited experimental control over transformation or transfection of cells, is frustrating. Any innovation in this area has the potential to radically influence the rate of progress in gene therapy. For example, it may be to change a cancer-like phenotype, to a benign one, or develop new strains of bacteria with useful properties. The figure above shows cells transfected with genes expressing green and red versions of GFP!
Dr. Casey explained the limitations of current electroporation technology, in which plasmid like DNA molecules are transferred largely by chance and brute force, through electrostatic "holes" punched into the plasma membrane. This technology and the complementary chemo-transfection methods have underpinned our understanding of genes and their expression in human cells for the last three decades. However, as interdisciplinary funding matures, more opportunities are emerging to bring greater levels of experimental control to these methods. In collaboration with Professor Cooper's group at Glasgow, Dr. Casey demonstrated how combining the laser technology developed for "cell-surgery" with the lab-on-a-chip work in Glasgow, was yielding good progress in reducing the risk to cell physiology associated with conventional electroporation, and offers the prospect of a more controlled approach to addressable delivery of macromolecules such as DNA for both research based genetic intervention and possibly therapeutic intervention in the future.Not surprisingly perhaps, as is the tradition at the Widnes Sci Bar (an audience that any Scientist should never underestimate!), Dr. Casey fielded questions relating to the laser technology and importantly the physical arrangement of the instrumentation to help appreciate the practicalities and the scale of these emerging technologies. In addition it should be noted that there are considerable challenges in "handling" nucleic acids, by whatever means, in view
I'll update the post with some Open Access supporting articles over the weekend! And....as always, these are my own views and comments, queries corrections are always welcome and never ignored!
Glossary of Terms
HeLa cells: experimental human derived cell line
Silica beads: micro scale spherical beads used in separation science, that can be coated with defined chemistries
Genome: the full complement of genes required for faithful copying of a cell or organism
Karyotype: the complement and morphology of the set of chromosomes in a eukaryotic cell. The number and gross structure of the microscopically examined chromosomes often reveal abnormalities which may be linked to certain genetic diseases
Membrane: the physical boundary around a cell. In animal cells it comprises hospholipids in a bilayer, interspersed with protein molecules
Green fluorescent protein (GFP): A naturally occurring protein isolated from jelly fish, that fluoresces bright green in natural light. It is used as a reporter for many cell functions
Transformation: the natural or experimental process of changing the characteristics of a bacterial cell by the addition of genetic material, usually DNA
Transfection: the equivalent of transformation in eukaryotic cells
Ribonucleic acid (RNA): the nucleic acid that contains the code for (usually) single proteins in all cells. It comes in a number of forms which are currently the focus of a great deal of research interest
