Of Twinkling Nanostars and the Possible Application of Stroboscopes in Biological Imaging

Imagine a strong crowd, as you see in a Manchester United versus Liverpool football match and you wished to concentrate on a particular person. How would you do it? Make him wear a fluorescent shirt and dye his hair (don’t do it in the middle of the crowd, I can’t guarantee your safety).

Purdue University researchers have been successful in focusing at the cell of interest among a background of equally noisy and boisterous biomolecules and other metabolically active cells. Currently, researchers use immunological techniques to create an antibody to a molecule and then visualize the ‘molecule of interest’ by tagging the antibody to a radioisotope or a fluorescent dye; and flow cytometry can sort out different types of cells.

The Purdue University team used gold coated nanoparticles with an iron oxide core that was impregnated in the cell they wished to see. They then subjected the specimen to a periodically changing magnetic field. The superparamagnetic cores (superparamagnetic nanoparticles have no net magnetization, but an external magnetic field can magnetize them) responded by rotating as the magnetic field rotated around them. The rotation could be seen in the ‘near infra-red’ light spectrum, as the incident light bounced off (scattered) the specially designed arms of the gold nanostar as it revolved. The rate (rpm) of this gyromagnetic (gyros means to rotate) twinkling could be externally controlled by varying the rate of the externally applied field. You now could identify the cell by its characteristic ‘twinkling’ (lighthouse type) effect.

I am tempted to go beyond what’s been achieved so far. Here I go. I guess you are all familiar what happens to the rotating ceiling fan blades when you turn on a fluorescent lamp. Don’t you see a momentary snapshot of the three blades (some have 4)? That’s what where stroboscope comes in. It consists of a Xenon lamp (ordinary fluorescent lamps could do, but incandescent lamps won’t work as the glowing filament takes time to extinguish) flashing at a controllable rate. The electronic circuitry may be had here.

Suppose that the fan is revolving at 1200 RPM and it is not changing. Set your stroboscope to flash at this rate. You’ll ‘see’ that the fan blades are absolutely not moving, which is certainly not true! But be there any mechanical defect in the fan, it will stand out as the centrifugal force widens it (provided that the fault is more or less tangential to the axis of rotation). Here also we are looking at our object of interest, aren’t we?

Now lets look what implication it might have in biological imaging. We now know that the gamma subunit of mitochondrial F type ATP Synthase ‘actually’ rotates when it is synthesizing ATP (reverse rotation occurs when ATP is hydrolyzed). There are other locomotive units within the cell as well. They comprise of actin and myosin based molecular motors. Could we study them using an externally adjustable stroboscope? The optical (electromagnetic) signals so obtained may then be similarly broken down into simpler trigonometric (sine and cosine) functions by Fourier analysis (Fourier transform) as was done in the ‘twinkling nanostars’ experiment. At least, we expect to get rid of some 'noise' and some good still photos. But if we wanted better resolution and used higher frequency (electromagnetic) for it, some extraneous error will be introduced. It's a trade-off!

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Reference: hyper-links, unless specifically mentioned
Principles of Biochemistry, Lehninger, 4th ed
http://en.wikipedia.org/wiki/ATP_synthase
Wei, Q., Song, H., Leonov, A., Hale, J., Oh, D., Ong, Q., Ritchie, K., & Wei, A. (2009). Gyromagnetic Imaging: Dynamic Optical Contrast Using Gold Nanostars with Magnetic Cores Journal of the American Chemical Society, 131 (28), 9728-9734 DOI: 10.1021/ja901562j

Molecular Motors

There are miniature machines in the cells of our bodies, which carry molecules on their 'legs' and walk on their 'heads', as they move from one part of the cell to the other. This way, they transport synaptic vesicles, small bags filled with neurotransmitters, from the nerve cell-body to the end of the neuron, the synaptic knobs. They also pull the chromosomes apart during cell division: mitosis and meiosis. In can also transport molecules from the synaptic knobs towards its cell body (retrograde transport). In this mode of transport, the nerve terminals pick up molecules (by endocytosis) such as nerve growth factors and send them to the cell-body. By this mechanism, they also pick up numerous viruses (polio virus that cause poliomyelitis etc.), toxins (toxins of Clostridium tetani; of tetanus) and send them to the interior as well, to its own detriment.

Broadly speaking, the molecular motors are of two types: those which move over actin (a filamentous molecule) and those which walk on microtubules. Kinesin is one such molecule which walks on microtubules. It has two heads. One head binds to ATP molecules (the energy currency of the cells) and hydrolyzes it to derive energy; while the other head bends and 'swings' forward. This way they produce a seemingly continuous motion. The cargo is bound to the other end (so lets we call them legs). Kinesins, with some exceptions, typically transport molecules towards the + end (polymerizing end) of microtubules. Dyneins, are quite like kinesins, but they carry molecules towards the -ve end of microtubules. It is of two types: cytoplasmic dynein and axonemal dynein. Axonemal dyneins are found in the cilia or flagella of cells, allowing the cell to move about by 'beating'.

Myosin on the other hand, walks along actin microfilaments. When we move our muscles, one of its heads hydrolyzes ATP molecules, and derives energy from it. It harnesses this energy to 'bend its other head', in what we call a power stroke. Thus, in doing so, it brings actin microfilaments closer (via this power stroke). Your muscle contracts, as a result. This is known as the 'ratchet theory' or walk along theory (of muscle contraction).

This video clip beautifully illustrates how kinesin does a perfect 10:

Walk like an Egyptian!

When we move from one place to another, we use our legs. So do all other animals. Movement (locomotion, to be precise) typically involves a support phase or stance phase when we support one foot on the ground and a swing phase when we swing the leg forward. Even our internal machineries also carry molecules or organelles from one part of the cell to other, by 'walking' in this fashion, and not by rolling.

Compare this (natural movement) with that of man made things that move. How the ancient Egyptians built the Pyramid in those days, is still debated. But they probably carried those heavy stones, on logs acting as wheels. Consider locomotives for example. Vehicles run on wheels, which rotate around an axis. Nearly all machines operate by rotating motors. Why then in nature, the transportation is looked after by the process of stepping?

One explanation is that in animal kingdom, a revolving appendage would mean detachment of that appendage, due to anatomic constraints. Secondly, devising an engine that would move in a step-like manner is not easily feasible due to the inherent unsymmetrical pull on its 'limbs' due to gravity, but the 'pi' factor would let the wheel move smoothly-in a continuous fashion.

I intend to write about the movement of 'molecular motors' in a later post. In the mean time it is really worth pondering why in nature things 'walk like an Egyptian'.

Microtubules: The Cytoskeletal Rail-Road

The cells have a rich cytoskeletal network, consisting of microfilaments, microtubules, intermediate filaments, and proteins that hold them to the interior of the cell. These cytoskeletal structures subserve various functions such as giving the cells its shape and allows small organelles inside the cells to go to other places in the cells. For example, in a nerve cell, the synaptic vesicles, which contain neurotransmitters, are synthesized in the cell bodies. These neurotransmitter filled vesicles are then transported by 'molecular motors' form the cell body to the synaptic knobs, situated at the other end of the neuron.

Not only do they (microtubules) serve as a 'rail line' on which molecular motors move (walk), they also act as 'traction wires' which pull chromosomes apart during cell division (mitosis). Thus in cancer, when cell division is excessive, drugs which interfere with microtubule formation (polymerization) can be employed effectively to curb this cell replication gone haywire. Vinca alkaloids (obtained from the Vinca rosea plant) such as vincristine (used in Hodgkin's Lymphoma, small cell lung cancer etc.), vinblastine (used in ALL or acute lymphoblastic leukemia in children), vinorelbine (used in NSCLC or non small cell carcinoma of the lung); and taxanes such as taxol (paclitaxel, used for the treatment of cancers of the breast and ovary) extracted from the western yew tree, are molecules which stabilize microtubules and thus arrest cell division.

Microtubules are composed of smaller subunits made of proteins, alpha tubulin and beta tubulin. Alpha and beta tubulins together form a heterodimer (hetero since alpha and beta tubulins are different molecules). 13 such hetero dimers join to form a ring and many such rings are joined in a stacked manner to form this hollow structure called microtubules. Microtubules are dynamic structures with one end forming by polymerization (+end) and the other end destroying by depolymerization (-end), as this 10 second video clip illustrates.