I Contain Multitudes

Our bodies are a genetic patchwork, possessing variation from cell to cell. Is that a good thing?

Olena Shmahalo for Quanta Magazine

Even healthy brains harbor genetic diversity, though scientists disagree over the extent.

Your DNA is supposed to be your blueprint, your unique master code, identical in every one of your tens of trillions of cells. It is why you are you, indivisible and whole, consistent from tip to toe.

But that’s really just a biological fairy tale. In reality, you are an assemblage of genetically distinctive cells, some of which have radically different operating instructions. This fact has only become clear in the last decade. Even though each of your cells supposedly contains a replica of the DNA in the fertilized egg that began your life, mutations, copying errors and editing mistakes began modifying that code as soon as your zygote self began to divide. In your adult body, your DNA is peppered by pinpoint mutations, riddled with repeated or rearranged or missing information, even lacking huge chromosome-sized chunks. Your data is hopelessly corrupt.

Most genome scientists assume that this DNA diversity, called “somatic mutation” or “structural variation,” is bad. Mutations and other genetic changes can alter the function of the cell, usually for the worse. Disorderly DNA is a hallmark of cancers, and genomic variation can cause a suite of brain disorders and malformations. It makes sense: Cells working off garbled information probably don’t function very well.

Most research to date has focused on how aberrant DNA drives disease, but even healthy bodies harbor genetic disorder. In the last few years, some researchers report that anywhere from 10 to 40 percent of brain cells and between 30 and 90 percent of human liver cells are aneuploid, meaning that one entire chromosome is either missing or duplicated. Copy number variations, in which chunks of DNA between 100 and a few million letters in length are multiplied or eliminated, also seem to be widespread in healthy people.

Scientists Create Simple Artificial ‘Cell’ Capable Of Spontaneous Movement

photo credit: Vesicle shapes created through osmosis. Image credit: Christoph Hohmann, Nano Initiative Munich

The cells that make up all living things are in constant interaction with their environment. Most cells perform complex chemical processes to ensure the cell and the organism remain healthy. Scientists have not yet been able to replicate a fully-functional synthetic cell, but it now appears they are off to a good start. A team of biophysicists have developed basic artificial vesicles capable of changing shape and moving spontaneously. 

The vesicles created in this study will be used in future design of increasingly-complex artificial cellular structures, capable of interacting with the environment and carrying out the same processes as a natural biological cell. The research was led by Andreas Bausch from Technische Universität München, and the paper appeared on the cover of Science.

Bausch’s team went back to the basics of cell biology and used biomolecules to build the most fundamental cellular structures from the protein level up. A rudimentary cytoskeleton was constructed by adding tiny tube-shaped polymers called microtubules inside the lipid bilayer membrane that served as the vesicle’s casing. Proteins called kinesins were also added to push along the microtubules, providing movement. Kinesins require coenzyme adenosine triphosphate (ATP) to function, which was added as a source of fuel.

Within the vesicle, the microtubules formed a constantly moving flat layer of liquid crystal. Liquid crystal is a state of matter that is neither liquid nor solid, yet has properties similar to both states. 

"One can picture the liquid crystal layer as tree logs drifting on the surface of a lake," lead author Felix Keber said in a press release. "When it becomes too congested, they line up in parallel but can still drift alongside each other.”

As the 2D arrangement of the microtubule liquid crystals are trying to completely line a 3D spheroid, it cannot be done flawlessly. Think of it like trying to gift wrap a basketball. The paper can pressed mostly flat, but eventually there will be unavoidable creases and faults. The faults within the microtubules caused some to be packed in a different orientation to fit in. 

The microtubule faults had not impacted the shape or integrity of the vesicle under normal circumstances, but that changed when the vesicles were subjected to different environmental conditions. As water was extracted from the vesicle due to osmosis, the bilayer membrane deflated. Movement from the microtubules in the faults caused the sagging membrane to adopt new shapes, including some with spike-like protrusions.

"With our synthetic biomolecular model we have created a novel option for developing minimal cell models," Bausch states. "It is ideally suited to increasing the complexity in a modular fashion in order to reconstruct cellular processes like cell migration or cell division in a controlled manner. That the artificially created system can be comprehensively described from a physical perspective gives us hope that in the next steps we will also be able to uncover the basic principles behind the manifold cell deformations."

ORIGINAL: IFLScience

by Lisa Winter

September 5, 2014

World-first working eukaryotic cell made from plastic

Researchers at the Institute for Molecules and Materials at Radboud University Nijmegen used a water droplet as the structure upon which they built the first polymer cell

Previously, chemists have managed to create artificial cell walls and developed synthetic DNA to produce self-replicating, synthetic bacterial cells. Now, for the first time, researchers have used polymers to produce an artificial eukaryotic cell capable of undertaking multiple chemical reactions through working organelles.

Eukaryotic cells are the building blocks for complex life-forms like plants and animals. The main distinction between the simpler and more ancient prokaryotic cells and eukaryotes is the presence of organelles in the latter. Organelles are specialized subunits within a cell that have a specific function, and which allow cells to undertake multiple chemical processes in an extremely small space.

This compartmentalization was one of the key features developed by nature during the early evolution of early life on Earth. It is also of interest to chemists as eukaryotic cells are capable of efficient chemistry at a very small scale, something which is difficult to replicate in the lab. That might be all about to change now chemists at Radboud University Nijmegen in The Netherlands have built the world’s first eukaryotic cell using plastic.

“Competing groups are working closer to biology; making cells from fatty acids, for example. We would like to do the same in the future," says Professor Jan van Hest who created the organelles with his PhD candidate Ruud Peters. "Another step would be to make cells that produce their own energy supply."

The researchers used a water droplet as the structure upon which they built the polymer cell. To create the organelles, they produced tiny polystyrene-b-poly spheres filled with enzymes designed to undertake set chemical processes. These sub-micrometric nanoreactors were then encapsulated in a coating of a polymer called polybutadiene-b-poly polymersome using emulsion-centrifugation to form a cell wall.

This formed a compartmentalized structure resembling nature’s eukaryotic cell. Within this, a multistep chemical reaction was undertaken which resembled a natural enzyme pathway. Using fluorescence, van Hest and Peters were able to show a chain of chemical reactions within the cell, proof they had created a polymer cell with working organelles.

"We are also working on ways of controlling the movement of chemicals within the cell, towards organelles," says van Hest. "By simulating these things, we are able to better understand living cells. One day we will even be able to make something that looks very much like the real thing."

Their work was published in the journals Angewandte Chemie and highlighted in Nature Chemistry.

Source: Radboud University Nijmegen

ORIGINAL: GizMag

By Grant Banks

January 15, 2014

A unique covalent bond in basement membrane is a primordial innovation for tissue evolution

Significance

The evolution of multicellular animals from single-celled ancestors was one of the most significant transitions of life on earth. The emergence of larger, more complex animals able to resist predation and colonize new environments was enabled, in part, by a collagen scaffold, which anchors cells together to form tissues and organs. Here, we show that a unique chemical bond, a link between sulfur and nitrogen atoms called a sulfilimine bond, arose over 500 Mya, binding this scaffold together and enabling tissues to withstand mechanical forces. Peroxidasin forms the bond by generating hypohalous acids as strong oxidants, a form of bleach, which normally function as antimicrobial agents. These understandings may lead to approaches for targeting tumors and treatment of other diseases.

Fig. 1.  The sulfilimine bond stabilizes collagen IV scaffolds by the cross-linking of triple helical building block protomers.
(A) The sulfilimine bond cross-links Met93 and Hyl211 at the interface between the trimeric NC1 domains of two adjoining protomers, forming a globular hexamer structure.
(B) Dimeric subunits reflect the presence of the sulfilimine bond in human collagen IV by immunoblot (JK2 Ab) and protein stain.
(C) MS analysis of tryptic peptides derived from dimeric subunits verified the presence of the bond by a mass difference of 2.0299 between theoretical mass of uncross-linked and observed mass of cross-linked peptides and subsequent multistep CID fragmentation (MS2/MS3) analyses.

Fig. 2. Multiple sequence alignment of collagen IV NC1 domains encompassing Met93 and Hyl211 amino acid residues and Pxdn among 11 metazoan and 1 protozoan phyla.
(A) Met93 and Lys/Hyl211 (yellow) are conserved in all eumetazoans, except for the cnidarian H. magnipapillata, and they are absent in the phyla of Placozoa and Porifera and the protozoan phylum Choanozoa. All sequences belong to the collagen IV α1-like subfamily of chains, except for Drosophila (viking) and Ascaris (α2 chain).
(B) Schematic representations of Pxdn. Pxdn sequence was incomplete on both ends for Mytilus, Clytia, Trichoplax, and Monosiga and short on one end for Saccoglossus, which is indicated here by a shortened schematic representation. Sequence data were gathered from *National Center for Biotechnology Information Reference Sequence, †gathered from whole-genome shotgun/transcriptome shotgun assembly, §generated by RNA-Seq analysis of animal tissues, or ¶assembled from cDNA libraries. All National Center for Biotechnology Information GenBank accession numbers are listed in Table S1.

Fig. 3. NC1 hexamers were excised from animal basement membranes and analyzed by SDS/PAGE as shown in Fig. 1 A and B. The dimeric subunits, which indicate the presence of the bond, were found in nine major eumetazoan phyla. Among eight cnidarians investigated, only Hydra NC1 lacked dimeric subunits. All NC1s were immunoblotted against the rat monoclonal antibody, JK2, except for C. elegans (rabbit polyclonal; NW-154) and Drosophila (mouse monoclonal; 6G7). Black outlines indicate the locations of cropping for blot images. 

Fig. 4. Expression of collagen IV and Pxdn during development in zebrafish and morpholino (MO) knockdown of peroxidasin in zebrafish embryos. (A) Pxdn and collagen IV expression during zebrafish embryonic development. Real-time qPCR studies were conducted to examine expression levels of Pxdn, collagen4α1, and collagen4α2. *Student t test P value < 0.03 compared with expression at 1,000 cells. Error bars = SEM. Blue, pxdn; red, col4a; black, col4a2. (B) Control and (C) Pxdn MO groups. MO-injected embryos displayed (D) general severe defects that include cardiac edema, smaller eyes, and gross trunk patterning defects (4/45), (E) partial curved trunk (21/45), or (F) normal development (20/45). (G) SDS/PAGE analysis of Pxdn MO embryonic phenotypes at 24 hpf by Western blot. Collagenase digests were normalized for total protein load by protein stain with SYPRO-Ruby (Fig. S8).


Abstract

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad—a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Footnotes

1A.L.F., R.M.V., and S.V.C. contributed equally to this work.

2A list of The Aspirnaut coauthors can be found in Table S2. Aspirnaut is a K--20 Science, Technology, Engineering, and Math (STEM) pipeline program for diversity that partners the experiential and content expertise of Vanderbilt University with rural kindergarten through 12th grade schools and diverse high school, undergraduate, and graduate students.

3To whom correspondence should be addressed. E-mail: billy.hudson@vanderbilt.edu.

Author contributions: R.M.V., S.V.C., V.K.P., V.P.Y., M.T.I., J.K.H., and B.G.H. designed research; A.L.F., S.V.C., G.B., V.P.Y., C.L.S., K.L.R., W.H.M., T.A.C., D.-B.B., R.E.S., and T.A. performed research; G.B. contributed new reagents/analytic tools; A.L.F., R.M.V., S.V.C., V.K.P., V.P.Y., D.-B.B., and R.E.S. analyzed data; and A.L.F. and B.G.H. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. GAMX01000001, GAMX01000002, GAND01000001, GAND01000002, GANB01000001, GANB01000002,GAMY01000001, GAMY01000002, GANA01000001, GANA01000002, GAMZ01000002, and GANC01000002).

This article contains supporting information online at 

www.pnas.org/lookup/suppl/doi:10.1073/pnas.1318499111/-/DCSupplemental.

Freely available online through the PNAS open access option. (Full Text)

ORIGINAL: PNAS

Aaron L. Fidlera,1, 

Roberto M. Vanacorea,b,1, 

Sergei V. Chetyrkina,b,1, 

Vadim K. Pedchenkoa,b,

Gautam Bhavea,b, 

Viravuth P. Yinc, 

Cody L. Stothersa, 

Kristie Lindsey Rosed,e, 

W. Hayes McDonaldd,e,f,

Travis A. Clarkg, 

Dorin-Bogdan Borzaa,b, 

Robert E. Steeleh, 

Michael T. Ivyi, 

The Aspirnautsb,2,

Julie K. Hudsonj, and 

Billy G. Hudsona,b,c,d,f,k,l,3

Author Affiliations

Edited* by Mina J. Bissell, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved November 22, 2013 (received for review September 30, 2013)

Researchers Japan find damage-free way to observe internal cell structures

Japanese scientists say they have developed the world’s first method to observe a live cell without damaging its internal structure.

The researchers said the procedure, using free-electron X-ray laser technology, will help advance an understanding of intracellular phenomena, such as the mechanism of cell division.

“By further improving performance, we will be able to take a look at smaller objects, as well as make closer observations of them,” said Yoshinori Nishino, an electronic science professor at Hokkaido University, who led the research team.

The team used the SACLA state-of-the-art X-ray facility in Hyogo Prefecture to expose bacteria, each 600 nanometers long, to a single dose of X-ray for 100-trillionth of a second. A nanometer is one-billionth of a meter.

An image taken at the SACLA X-ray facility in Hyogo Prefecture reveals a live cell’s internal structures. (Provided by Yoshinori Nishino)

It has been impossible to observe live cells using a conventional X-ray device because they become severely damaged. The new method also eliminates the need to fix cells’ internal structures with resin and stain them when using electron microscopy techniques.

The SACLA facility, set up by the RIKEN research institute and others at a cost of 39 billion yen ($372 million), enabled Nishino’s team to capture an image of bacteria almost free from damage.

According to the scientists, substances that appeared to be a gathering of DNA, the molecule that encodes genetic information, could be observed in the cells.

The findings were published in the British scientific journal Nature Communications on Jan. 7.

ORIGINAL: AJW

By JIN NISHIKAWA/ Staff Writer
January 08, 2014

Video: Virus caught in the act of infecting a cell

ORIGINAL: LabSpaces

Friday, January 11, 2013

Researchers found that the T7 virus has six tail fibers that are folded back against its capsid. The fibers extend as the virus locates a suitable host and as it “walks” across its host cell surface to find a site to infect. Credit: From Hu et. al. 2013. Science Express.

The top images are tomograms of the virus in action. The illustrations show T7 using its fibers to “walk” across the cell surface and infect the cell. Credit: From Hu et. al. 2013, Science Express.

The detailed changes in the structure of a virus as it infects an E. coli bacterium have been observed for the first time, report researchers from The University of Texas at Austin and The University of Texas Health Science Center at Houston (UT Health) Medical School this week in Science Express.

To infect a cell, a virus must be able to first find a suitable cell and then eject its genetic material into its host. This robot-like process has been observed in a virus called T7 and visualized by Ian Molineux, professor of biology at The University of Texas at Austin, and his colleagues.

The researchers show that when searching for its prey, the virus briefly extends — like feelers — one or two of six ultra-thin fibers it normally keeps folded at the base of its head.

Once a suitable host has been located, the virus behaves a bit like a planetary rover, extending these fibers to walk randomly across the surface of the cell and find an optimal site for infection.

At the preferred infection site, the virus goes through a major change in structure in which it ejects some of its proteins through the bacterium's cell membrane, creating a path for the virus's genetic material to enter the host.

After the viral DNA has been ejected, the protein path collapses and the infected cell membrane reseals.

"Although many of these details are specific to T7," said Molineux, "the overall process completely changes our understanding of how a virus infects a cell."

For example, the researchers now know that most of the fibers are usually bound to the virus head rather than extended, as was previously thought. That those fibers are in a dynamic equilibrium between bound and extended states is also new.

Molineux said that the idea that phages "walk" over the cell surface was previously proposed, but their paper provides the first experimental evidence that this is the case.

This is also the first time that scientists have made actual images showing how the virus's tail extends into the host — the very action that allows it to infect a cell with its DNA.

"I first hypothesized that T7 made an extended tail more than 10 years ago," said Molineux, "but this is the first irrefutable experimental evidence for the idea and provides the first images of what it looks like."

The researchers used a combination of genetics and cryo-electron tomography to image the infection process. Cryo-electron tomography is a process similar to a CT scan, but it is scaled to study objects with a diameter a thousandth the thickness of a human hair.

###

University of Texas at Austin: http://www.utexas.edu

Thanks to University of Texas at Austin for this article.

The origins of the first immortal human cells

ORIGINAL: ScienceDump

by Jur on

11/14/2012

In 1951, a scientist at Johns Hopkins Hospital in Baltimore, Maryland, created the first immortal human cell line with a tissue sample taken from a young black woman with cervical cancer. Those cells, called HeLa cells, quickly became invaluable to medical research—though their donor remained a mystery for decades.

The first immortal human cells grown in culture, they are still alive today, though the donor has been dead for more than sixty years. If you could pile all HeLa cells ever grown onto a scale, they’d weigh more than 50 million metric tons—as much as a hundred Empire State Buildings.

HeLa cells were vital for developing the polio vaccine; uncovered secrets of cancer, viruses, and the atom bomb’s effects; helped lead to important advances like in vitro fertilization, cloning, and gene mapping; and have been bought and sold by the billions.

Photo of the donor: Henrietta Lacks and her husband

Imaging the Sustainable Cell

ORIGINAL: Cell Press

Register for a Free Webinar Imaging the Sustainable Cell  Live date: Thursday, September 27, 2012 12:00pm EDT 9am PDT, 5pm BST   This webinar is for any biologist interested in learning about how cells balance resources and make the decision between redirecting resources and initiating death. It will be a great opportunity to learn how the experts are studying these processes and how you can monitor and image each process. About this webinar: Sustainability is a hot topic. Disposing of and recycling what we no longer need is critical, and this is true down at the cellular level as well. Pathways and processes across biology connect to at least one of the cell's sustainability programs, which include the ubiquitin-proteasome pathway, autophagy, and cell death. Find out how your work fits into the picture in this webinar, where Doug Green, Ana Maria Cuervo, and Ivan Dikic will discuss some of their recent findings and how they look for signs of sustainability (or lack thereof). Click on the link below to register http://view6.workcast.net/register?pak=9930857197891551&referrer=text Unable to join us on September 27th? This webinar will be archived for 6 months for on-demand viewing. Register today to ensure you receive updates on how to gain access after the live event! Be sure to check out our archive of past webinars athttp://www.cell.com/webinars Image credit: Laura Santambrogio Moderated by: Sabbi Lall Scientific Editor, Cell Reports Speakers: Douglas Green St Jude Children's Research Hospital Ana Maria Cuervo Albert Einstein College of Medicine Ivan Dikic Goethe University Supporting Journal: Cell Reports papers related to the webinar can be found in this Cell Reports Collection. To unsubscribe, please click here