4 day old zebrafish embryo labelled with SV2 and acetylated tubulin antibodies showing axon tracts(green) and neuropil(red)viewed from lateral and dorsal orientations.

I TA for a course that involves photographing zebrafish embryos through development, and it is one of the most incredible things I’ve ever looked at. This image was taken by scientists at UCL in London. 

Crepidula fornicata veliger larvae | ZEISS Microscopy

Confocal image (extended focus Z stack) of a Crepidula fornicata (slipper limpet) veliger larva. Stained with phalloidin (F-actin; purple), DAPI (cell nuclei, blue), anti-serotonin (yellow), and anti-acetylated tubulin (red).

The shell image was created from the DIC picture collected during the confocal scan. The C-shaped line of nuclei are cells at the edge of the velum; the acetylated tubulin staining reveals the ciliated surface of the velum. The F-actin staining highlights the main larval retractor muscle. Serotonin reveals the serotonergic neuron cell bodies and axons. 



The Penrose-Hameroff theory of orchestrated objective reduction (Orch OR) postulates quantum computation in microtubules inside brain neurons underlying consciousness. Specifically, Orch OR proposes that tubulin proteins comprising microtubule cylindrical lattices function as ‘bits’ – switching between alternative states (e.g. of 1 or 0), as well as quantum bits or 'qubits’ (existing transiently as quantum superposition of both 1 AND 0). Despite increasing evidence for functional quantum effects in warm biological systems, Orch OR has been recently criticized, e.g. in Phys Rev E by McKemmish et al (2009), who claim the nature and energetic requirements for switching of tubulin bits and qubits in microtubules make Orch OR biologically unfeasible and unsalvageable irrespective of any conceivable modification. Here we show that McKemmish et al misrepresent tubulin bit switching as proposed in Orch OR, and merely disprove their own misrepresentation. Specifically we address their allegations regarding regulation of tubulin switching by 1) van der Waals London forces, 2) GTP hydrolysis and 3) Fröhlich coherence, and show how they are wrong on all counts. We clarify certain aspects of tubulin with regard to potential bit/qubit function, and describe topological tubulin qubits specific to microtubule geometry with particular reference to helical ballistic conductance discovered by Bandyopadhyay. Orch OR remains viable and testable.

How to Make Transgenic Flies in 3 Easy Steps (or Crosses)

     As promised, I will explain the method of creating the aforementioned strain of flies. Just a disclaimer- the process is genetics-heavy; there’s not exactly a way to explain it thoroughly without delving into the technicalities, but I hope that my description is not too cluttered. Personally, I find the process itself highly fascinating; the techniques scientists have devised to manipulate flies and generate desired characteristics are so vast and so precise it’s insane.

     One of the mechanisms of creating novel strains, which I mentioned earlier, is the transposable element.  These elements are basically jumping sequences of DNA that move around within a genome, landing wherever they please. Certain transposable elements, such as “P” elements for example, have a high tendency to land near a “TATAA box” controlling a the expression of a certain gene. TATAA boxes are basically RNA polymerase binding sites; they are sequences of DNA that signal to RNA polymerases where to begin transcription. P elements tend to land right near these “TATAA” sequences, around the start of a specific gene. Say two genes are far apart from each other, P elements almost never land in the gene desert between them- they insert themselves close to the start of one of the genes. Although P elements can be predicted to land near TATAA boxes, there is no way to tell WHICH TATAA box they will land near. There are many genes on a single chromosome, and P elements have an equal chance on landing on any one.

     Piggybac, on the other hand, has a truly random insertion pattern. At times, Piggybacs insert themselves in gene deserts, or at the ends of genes, in fact Piggybacs sometimes land in the middle of genes, disrupting the sequence of the gene and its expression.

     The fly embryos into which I plan to insert my DNA plasmids in order to make transgenic flies automatically produce Piggybac transposases, (conveniently) the type of transposable element that I want to use. This makes my life much easier – I don’t have to manually mix the transposase plasmid with my DNA plasmid, because the Piggybac automatically produced by the embryo will bind with my DNA plasmid.

     So, I’ll start with my first generation of flies. These flies have the following genotype:

     Note: Fruit flies have four pairs of chromosomes- three autosomal pairs, and one X/Y pair. While reading Drosophila genotypes, the main thing to keep in mind is that the semicolons separate the different chromosomes. For example, the above genotype indicates that “w,” an eye color gene, is on the first chromosome, and that SP-1 and CurlyO are both on the second. The “plus” signs in the male genotype represent wild-type chromosomes, or chromosomes with normal genes, and the“W-“ (white mutant) produces white eye color (a.k.a. no eye color). Sp-1 and CurlyO are both homozygous lethal mutations – this means that any fly that has two copies of either gene will not survive. Any of the offspring of this parent generation of flies is that inherits a genotype of CurlyO/CurlyO, or Sp-1/Sp-1 will die in their larval stage. Using a Punnett Square, it can be determined that roughly ½ of the offspring will have either one of these genotypes, so only ½ of the total offspring will survive. The good news though, is that 100% of these offspring will have the CurlyO chromosome that produces Piggybac transposase, so all of our flies will express Piggybac transposase.

     So I cross these flies (mate them), and wait for them to reproduce. Then, I will screen the progeny, or the offspring, of the flies I crossed, in order to select only the flies that have colored eyes (denoted by w+ genotype). These flies should also have the Piggybac transposase. Since the “w+” genes will be floating around in the “w-“ background, there will only be a fraction of the progeny that have the “w+” genes; only a subset of germ cells will actually take up the DNA we injected. 

     Now tubulin, whose promoter drives the expression of Piggybac, is a protein that is expressed in all cells, including eye and germ cells. Tubulin (along with the Piggybac transposase) will continue to move eye color genes around all over the fly, so the eye becomes mosaic colored, due to the constantly shifting “w+” gene. 

     Once these flies are chosen, I have to do a second cross. I cross above flies with males of the following genotype: (W-/Y;+/+;+/+)

     Out of the progeny of this cross, I can identify which flies have CurlyO because those flies will also have mosaic colored eyes from the Piggybac transposase. (CurlyO produces tubulin which drives the expression of Piggybac, so the two will always be together). If I select the flies from the progeny that do not have CurlyO, then there is a chance that they will have solid eye color. This solid colored fraction of flies will have “w+” genes amidst the “w-“ background, and they will have no Piggybac without the CurlyO.

     Now that I have these solid eye-colored flies, I will conduct more crosses using “balancer chromosomes” in order to find out which chromosome my Piggybac element actually landed on. Put simply, balancer chromosomes are genetic tools used to maintain heterozygous populations. For example, the Tm3 chromosome (a 3rd chromosome balancer that produces stubble hairs), is a balancer chromosome-any fly with two Tm3 chromosomes would not survive, since homozygous Tm3 is lethal. I could probably spend hours trying to explain more about how balancer chromosomes work and how useful they are; for anyone interested, I would suggest this article for a basic overview, and this book (starting on chapter 1 page 12) for a more detailed description. The reason I am using balancer chromosomes is to stabilize the fly populations I have created, so that I do not lose my injected Piggybac. Since my flies are heterozygous, it is possible that a portion of their offspring will be homozygous. Balancer chromosomes with homozygous lethal mutations allow me to eliminate flies with homozygous balancers, since they will die in their larval stages. Hence the only flies that survive from generation to generation are the heterozygous flies that contain my Piggybac element.

     Now to piece all these steps back into the larger scheme of things- these now stable populations are what I, and the rest of my class at Exeter, will use to locate the Piggybac element we initially injected. Ideally, most flies will have this element in a different location due to the random hopping of the Piggybac element, and we want to cover as many locations as possible! Like I had mentioned, if we are able to map exactly where our Piggybac elements land, and publish these molecular locations on Flybase, our lines can be used to study the functions of genes that our Piggybac elements disrupted, or to study cells that express these genes!

      I hope that all of this made sense, and if any part of it was messy, feel free to ask me about it via the “Questions?” tab at the top of the page!



(Image caption: TTLL7 (gold structures on the left) impacts cell function by binding to microtubules (silver structure made up of purple and yellow subunits) and adding chemical markers to the surface. Credit: Roll-Mecak lab)

Scientists unravel the mystery of the tubulin code

Driving down the highway, you encounter ever-changing signs— speed limits, exits, food and gas options. Seeing these roadside markers may cause you to slow down, change lanes or start thinking about lunch. In a similar way, cellular structures called microtubules are tagged with a variety of chemical markers that can influence cell functions. The pattern of these markers makes up the “tubulin code” and according to a paper published in Cell, scientists at NIH’s National Institute of Neurological Disorders and Stroke (NINDS) have uncovered the mechanism behind one of the main writers of this code, tubulin tyrosine ligase-7 (TTLL7).

“Understanding the structural characteristics of this specific molecule opens the door to learning how elaborate patterns of chemical markers are laid down on microtubules. Deciphering the tubulin code could tell us how the markers affect normal cellular function as well as what happens when they are damaged, which can lead to neurodegenerative disorders,” said Antonina Roll-Mecak, Ph.D., NINDS scientist and senior author of the study.

TTLL7 is a protein that adds glutamate tags onto microtubules. Using a number of advanced imaging and biochemical techniques, Dr. Roll-Mecak and her colleagues at the Scripps Research Institute in La Jolla, California, have revealed the 3-D structure of TTLL7 bound to the microtubule. There are nine proteins that make up the TTLL family, but TTLL7 is the most abundant in the brain and one of the main tubulin code writers. These results represent the first atomic structure of any member of the TTLL family.

The findings define how TTLL7 interacts with microtubules and how members of the TTLL family use common strategies to mark microtubules with glutamate tags. Dr. Roll-Mecak and her team were able to see how TTLL7 positions itself on the microtubule by grabbing onto the microtubule tails.

“This was a very surprising result, as no one had been able to visualize these tails on the microtubule before,” said Dr. Roll-Mecak.

Microtubules are cylindrical structures that provide shape to cells and act as conveyor belts, ferrying molecular cargo throughout cells. Although all microtubules have the same basic appearance, they are marked on their outside surface with a variety of chemical groups. These markers impact a cell’s activity by changing the stability of microtubules, thus affecting cell shape, or by repositioning molecular cargo traveling on the microtubules.

“The microtubule markers are constantly being added and removed, depending on the local needs of the cell. Think about a highway system where street signs are constantly changing and roads are quickly built or torn apart,” said Dr. Roll-Mecak.

The most common microtubule marker in the brain is glutamate. The addition of glutamate markers to microtubules plays important roles in brain development and brain cell repair following injury. For example, one of the signatures of damaged cells in cancer or blunt trauma is a change in the pattern of these microtubule markers. In addition, mutations in TTLL genes have been linked with several neurodegenerative disorders.

“Our detailed analysis of TTLL7 also may provide important insights into ways that the other members of the TTLL family function. This study is the first step in gaining a more complete picture of how the tubulin code is established,” said Dr. Roll-Mecak.

Her lab plans to extend their research by investigating interactions between members of this family of proteins. “We want to mix and match the TTLL proteins to see how we can control patterns of microtubule tagging. From that, we can learn how the cell is making those patterns and what happens during cellular damage, as in cancers or neurodegeneration, when these patterns are disrupted,” said Dr. Roll-Mecak.

She added that this research may lead to the development of small molecules that can regulate activity of TTLL proteins, which may have implications for disorders linked to mutations in TTLL genes.

NIH Scientists Unravel the Mystery of Cellular Traffic Cops

Driving  down the highway, you encounter ever-changing signs — speed limits, exits, food  and gas options. Seeing these roadside markers may cause you to slow down,  change lanes or start thinking about lunch. In a similar way, cellular  structures called microtubules are tagged with a variety of chemical markers  that can influence cell functions. The pattern of these markers makes up the  “tubulin code.” 

“Understanding  the structural characteristics of this specific molecule opens the door to  learning how elaborate patterns of chemical markers are laid down on  microtubules. Deciphering the tubulin code could tell us how the markers affect  normal cellular function as well as what happens when they are damaged, which  can lead to neurodegenerative disorders,” said Antonina Roll-Mecak, Ph.D.,  NINDS scientist and senior author of the study.  

Read More

This research was conducted by scientists at  National Institutes of Health’s National Institute of Neurological Disorders and Stroke (NINDS) of the NIH.

Cnidaria, MultiView Light Sheet Microscopy (3 of 4) by ZEISS Microscopy
Via Flickr:
Immunostaining of planktonic Cnidaria. Acetylated tubulin (green), myosin (red), nuclei (blue). Image taken with ZEISS Lightsheet Z.1 during the EMBO course on Marine Animal Models in Evolution &amp; Development, Sweden 2013. <a href=“http://www.zeiss.com/lightsheet” rel=“nofollow”>www.zeiss.com/lightsheet</a> Sample courtesy of Helena Parra, Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona.

Is it possible to predict electromagnetic resonances in proteins, DNA and #RNA?

Background: It has been shown that there are electromagnetic resonances in biological molecules (proteins, DNA and #RNA) in the wide range of frequencies including THz, GHz, MHz and KHz. These resonances could be important for biological function of macromolecules, as well as could be used in development of devices like molecular computers. As experimental measurements of macromolecular resonances are timely and costly there is a need for computational methods that can reliably predict these resonances.We have previously used the Resonant Recognition Model (RRM) to predict electromagnetic resonances in tubulin and microtubules. Consequently, these predictions were confirmed experimentally. Methods: The RRM is developed by authors and is based on findings that protein, DNA and #RNA electromagnetic resonances are related to the free electron energy distribution along the macromolecule. Results: Here, we applied the Resonant Recognition Model (RRM) to predict possible electromagnetic resonances in telomerase as an example of protein, telomere as an example of DNA and TERT #mRNA as an example of #RNA macromolecules. Conclusion: We propose that RRM is a powerful model that can computationally predict protein, DNA and #RNA electromagnetic resonances. http://bit.ly/1Q0exTu #BMC

Expanding the phenotypic spectrum and variability of endocrine abnormalities associated with TUBB3 E410K syndrome.

PubMed: Related Articles

Expanding the phenotypic spectrum and variability of endocrine abnormalities associated with TUBB3 E410K syndrome.

J Clin Endocrinol Metab. 2015 Mar;100(3):E473-7

Authors: Balasubramanian R, Chew S, MacKinnon SE, Kang PB, Andrews C, Chan WM, Engle EC

CONTEXT: A heterozygous de novo c.1228G>A mutation (E410K) in the TUBB3 gene encoding the neuronal-specific β-tubulin isotype 3 (TUBB3) causes the TUBB3 E410K syndrome characterized by congenital fibrosis of the extraocular muscles (CFEOM), facial weakness, intellectual and social disabilities, and Kallmann syndrome (anosmia with hypogonadotropic hypogonadism). All TUBB3 E410K subjects reported to date are sporadic cases.
OBJECTIVE: This study aimed to report the clinical, genetic, and molecular features of a familial presentation of the TUBB3 E410K syndrome.
DESIGN: Case report of a mother and three affected children with clinical features of the TUBB3 E410K syndrome.
SETTING: Academic Medical Center.
MAIN OUTCOME MEASURES: Genetic analysis of the TUBB3 gene and clinical evaluation of endocrine and nonendocrine phenotypes.
RESULTS: A de novo TUBB3 c.1228G>A mutation arose in a female proband who displayed CFEOM, facial weakness, intellectual and social disabilities, and anosmia. However, she underwent normal sexual development at puberty and had three spontaneous pregnancies with subsequent autosomal-dominant inheritance of the mutation by her three boys. All sons displayed nonendocrine features of the TUBB3 E410K syndrome similar to their mother but, in addition, had variable features suggestive of additional endocrine abnormalities.
CONCLUSIONS: This first report of an autosomal-dominant inheritance of the TUBB3 c.1228G>A mutation in a family provides new insights into the spectrum and variability of endocrine phenotypes associated with the TUBB3 E410K syndrome. These observations emphasize the need for appropriate clinical evaluation and complicate genetic counseling of patients and families with this syndrome.

PMID: 25559402 [PubMed - indexed for MEDLINE] http://dlvr.it/9pR0VB