March 12, 2014

I love a lot of things that are rings, especially donuts. Turns out, though, that ring chromosomes are terrible news. A recent paper shows the loss of ring chromosomes when cells are reprogrammed, suggesting possible ‘chromosome therapy’ through cell reprogramming.

Ring chromosomes form when the two arms of a chromosome fuse, and are sometimes associated with large terminal deletions. These ring chromosomes lead to birth defects, mental disabilities, and growth retardation. Unfortunately, there are no treatments for ring chromosome disorders due the severity of the aberrations. In a recent study, Bershteyn and colleagues generated induced pluripotent stem cells (iPSCs) from cells of a Miller Dieker Syndrome patient with large deletions on ring chromosome 17. The induced stem cells lost the ring chromosome and duplicated the normal homologous chromosome through a mechanism called compensatory uniparental disomy. The images above show two karyotypes—one with the ring chromosome 17 (left, inset), and one without (right).

Bershteyn, M., Hayashi, Y., Desachy, G., Hsiao, E., Sami, S., Tsang, K., Weiss, L., Kriegstein, A., Yamanaka, S., & Wynshaw-Boris, A. (2014). Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells Nature, 507 (7490), 99-103 DOI: 10.1038/nature12923
Adapted by permission from Macmillan Publishers Ltd, copyright ©2014

May 24, 2013

There isn’t a cell biologist out there who doesn’t fantasize about reaching her hands into a cell and physically manipulating whatever protein or structure that she obsesses over.  While we can’t do that with our own hands, optical tweezers can…and the information we learn is invaluable.  Today’s image is from a paper that uses optical tweezers to measure the forces within a mitotic spindle.

The mechanics within a mitotic spindle are complicated, and cannot be fully understood until all of the forces that exist within it are determined.  A recent paper measures the forces of chromosome and spindle pole movements using optical tweezers to trap either kinetochores or spindle poles.  Optical tweezers use refracted light to trap small objects within a cell, and are able to stop the movement of the object as the laser power of the trap approaches (or exceeds) the cell’s own force on the object.  With this technology, Ferraro-Gideon and colleagues measured the forces in mitotic spindles in several samples—a flatworm, a crane fly, and a mammalian spindle.  The force used to stop chromosome movement was closer to the theoretical values of chromosome force, much less than the force values calculated by experiments performed by Bruce Nicklas in the 1980s using needles to manipulate chromosome movement.  In the DIC (left) and fluorescence images (right) above, a trap (square) is applied to kinetochores in mammalian cells.  The trap did not damage the microtubules of the spindle (right image).

Ferraro-Gideon, J., Sheykhani, R., Zhu, Q., Duquette, M., Berns, M., & Forer, A. (2013). Measurements of forces produced by the mitotic spindle using optical tweezers Molecular Biology of the Cell, 24 (9), 1375-1386 DOI: 10.1091/mbc.E12-12-0901

Check out an extended report on these experiments, and others from this lab:
Sheykhani, R., Baker, N., Gomez-Godinez, V., Liaw, L., Shah, J., Berns, M., & Forer, A. (2013). The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle Cytoskeleton, 70 (5), 241-259 DOI: 10.1002/cm.21104

April 9, 2013

Our genome is chock full of so many things that aren’t even genes.  In fact, only about 2% of the human genome actually encodes protein sequences...mind blown, right?!  There are many different kinds of elements and domains within our genome that regulate gene expression through their roles in chromosome architecture and organization.  Today’s image is from a paper that describes the dynamics of one type of domain—the lamina associated domain.

The nuclear lamina is a protein layer that coats the inside nuclear membrane, and serves to anchor chromosomes.  Regions in the genome called lamina associated domains (LADs) specifically associate with the nuclear lamina.  LADs cover about 35-40% of the genome, suggesting that they may affect chromosome position and architecture.  A recent paper tracks LAD-nuclear lamina interactions throughout the cell cycle in single cells.  Kind and colleagues show that about 30% of LADs are positioned at the nuclear periphery.  LADs are stochastically positioned after mitosis, meaning that their position is not directly inherited.  In addition, these contacts are linked with gene expression and histone modifications.  In the images above, the nuclear lamina (blue) and mitotic spindle (red) are shown throughout the different stages of mitosis.  LADs (green) are rounded and at the nuclear periphery during prophase, and then become banded along chromosomes once the nuclear envelope breaks down (prometaphase and metaphase).  During cytokinesis, LADs are seen within the nucleus, but not yet positioned at the periphery.

Kind, J., Pagie, L., Ortabozkoyun, H., Boyle, S., de Vries, S., Janssen, H., Amendola, M., Nolen, L., Bickmore, W., & van Steensel, B. (2013). Single-Cell Dynamics of Genome-Nuclear Lamina Interactions Cell, 153 (1), 178-192 DOI: 10.1016/j.cell.2013.02.028
Copyright ©2013 Elsevier Ltd. All rights reserved.

February 15, 2013

The regulation of genes occurs at many levels, one of the first of which is physical access to a gene.  The way DNA is packaged may or may not allow transcription machinery from even getting to an area of the genome.  Understanding these modifications in each cell type and location is important, but difficult within a tissue or tumor of different cell types.  A recent paper tackles this problem with a new method.

Histones are proteins that package DNA to condense its size and to aid in gene regulation.  Histones can be modified in many different ways, and these modifications affect how accessible or inaccessible a certain region of the genome is to gene transcription.  Chromatin immunoprecipitation, the current technique to understand histone modifications at different gene sites, does not provide information on histone modifications of single cells within a complex environment of different cell types.  A recent paper describes a new technique that allows the visualization of histone modifications at single-cell resolution within a fixed tissue.  Using this technique, Gomez and colleagues tracked one specific histone modification, dimethylation of lysine 4 of histone H3 (H3K4me2) at the genetic loci for MYH11 in smooth muscle cells (SMC).  SMC-containing tissues contain other cell types, and even non-SMC tissues contain some SMCs due to vascularization.  In the images above, MYH11 H3K4me2 modifications (red, arrows) were found only in SMCs (green cells) in sections of human carotid artery tissue (DNA in blue).  Higher magnification images are bottom row.

Gomez, D., Shankman, L., Nguyen, A., & Owens, G. (2013). Detection of histone modifications at specific gene loci in single cells in histological sections Nature Methods, 10 (2), 171-177 DOI: 10.1038/nmeth.2332
Adapted by permission from Macmillan Publishers Ltd, copyright ©2013 

January 28, 2013

In my many hours in a dark microscopy suite, I would stare slack-jawed at cells going through mitosis like a perv at a peep show. Beneath the grace of this serious cellular rite of passage is a massive amount of regulation, which just adds to the fascination so many biologists have for the event. Today’s image is from a paper that describes the generation and importance of forces that help align chromosomes on a metaphase plate.

During mitosis, chromosomes must align on the metaphase plate with attachments to opposite spindle poles. Only after this precise alignment can the cell begin a cascade of signals and checkpoints to trigger anaphase, which segregates sister chromatids into their eventual daughter cells. Chromosome alignment gets a hand from polar ejection forces (PEFs), which are forces generated by the microtubule-based motor kinesin found on chromosome arms. These chromokinsesins (kinesin-10 family members) are thought to walk chromosome arms away from spindle poles along microtubules, and are important for timely congression of chromosomes to the metaphase plate. A recent paper describes the relationship between PEFs and the stability of spindle-chromosome attachments. Cane and colleagues manipulated the levels of the fruit fly kinesin-10 protein NOD to similarly manipulate the tension and forces applied to chromosomes. When both kinetochores of a chromosome pair were incorrectly attached to the same spindle pole (called a syntelic attachment), NOD stabilized the attachments by preventing error correction by the protein Aurora B. From their results, Cane and colleagues show that PEFs regulate the stability of kinetochore attachment to spindle microtubules. In the time-lapse images above, NOD overexpression in a fruit fly cell caused an abnormal metaphase plate to form. Despite the syntelic attachments of many chromosomes, the cell still entered anaphase (AO), resulting in daughter cells with too many nuclei (far right image). NOD signal is red, microtubules are green. 

BONUS!!  Beautiful movies from the paper can be found here.

Cane, S., Ye, A., Luks-Morgan, S., & Maresca, T. (2013). Elevated polar ejection forces stabilize kinetochore-microtubule attachments originally published in the Journal of Cell Biology, 200 (2), 203-218 DOI: 10.1083/jcb.201211119

July 30, 2012

It may appear that chromosomes are just floating around inside of the nucleus, but that couldn’t be further from the truth. Inside that double membrane of the nucleus is a lot of chromosome choreography, and this is especially true during meiosis. Today’s image is from a paper showing the interaction between two nuclear envelope proteins and their regulation of chromosome dynamics and pairing.

Meiosis is a type of cell division that reduces the number of chromosomes down to one copy of each in the resulting daughter cells, called gametes (eggs and sperm). During meiosis, the pairing of homologous chromosomes (matching chromosomes) is important for recombination and chromosome segregation later in meiosis. Recombination is the exchange of DNA regions between two paired chromosomes, and is key in generating genetic variation. In yeast and worms, KASH domain proteins and SUN domain proteins of the nuclear envelope interact to ensure proper chromosome pairing and positioning within the nucleus. Mammals were known to have a SUN domain protein, SUN1, and a recent paper identified its KASH domain binding partner, KASH5. Morimoto and colleagues found that KASH5 and SUN1 both localize at telomeres, regions at the ends of chromosomes, during meiosis in mice. In addition, KASH5 interacts with the microtubule-associated dynein-dynactin complex to regulate chromosome movement. In the images above, chromosomes from mouse spermatocytes have both KASH5 and SUN1 localized at telomeres throughout meiosis (chromosomes are blue).

Akihiro Morimoto, Hiroki Shibuya, Xiaoqiang Zhu, Jihye Kim, Kei-ichiro Ishiguro, Min Han, & Yoshinori Watanabe (2012). A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis originally published in the Journal of Cell Biology, 198 (2), 165-172 DOI: 10.1083/jcb.201204085

June 18, 2012

I love chromosomes. I am always in awe when I see these little things that are chock full of information and instructions for a cell, and in turn, a whole organism. When I think about how finely-tuned the dance is that allows chromosome segregation to happen correctly every time during mitosis, I am beyond impressed. Today’s image is from a paper describing how sister chromatids become bound to one another.

Sister chromatids remain held together until anaphase segregates them into two future daughter cells during mitosis. Chromatid cohesion is mediated by the cohesin complex of proteins and is established long before mitosis. A recent study identified the role of a protein called XEco2 in acetylating a cohesin complex member called Smc3, a step that is required for the establishment of chromatid cohesion. In addition, Higashi and colleagues found that this role of XEco2 is important prior to DNA replication, requiring the formation of the pre-replication complex. Later, DNA replication serves to stabilize cohesion between sister chromatids. In the images above, sister chromatids are bound together in control cells and in cells depleted of XEco1 (left two columns). In cell depleted of XEco2, XEco1 and XEco2 together, or cohesin (middle and right two columns), sister chromatids are not bound tightly to one another.

Higashi TL, Ikeda M, Tanaka H, Nakagawa T, Bando M, Shirahige K, Kubota Y, Takisawa H, Masukata H, & Takahashi TS (2012). The Prereplication Complex Recruits XEco2 to Chromatin to Promote Cohesin Acetylation in Xenopus Egg Extracts. Current biology : CB, 22 (11), 977-88 PMID: 22560615

Copyright ©2012 Elsevier Ltd. All rights reserved.

 

March 22, 2012

I love mitotic spindles, so of course I love early fruit fly development and its rapid, synchronized syncytial divisions. Watching these mitotic spindles perform a synchronized swimming routine, complete with tiny little swim caps and nose plugs, is always a treat. Today’s image is from a paper that helps to define the relationship between DNA replication, chromosome condensation, and mitotic progression.

During the cell cycle, DNA strands are replicated. After proper DNA replication, the very long strands are compacted in order to allow efficient and accurate separation of chromosomes during mitosis. When chromosome condensation doesn’t occur correctly, the progress through mitosis is disrupted. This may be due to the well-studied spindle assembly checkpoint, or there may be a checkpoint that monitors chromosome condensation. A recent paper describes results showing the effects of certain inhibitors on chromosome condensation and mitotic progression, marked by entry into anaphase. Fasulo and colleagues found that the inhibitors that severely disrupted chromosome condensation also disrupted anaphase onset. These delays occurred through disruption of the Wee1 kinase, and not due to the spindle assembly checkpoint. By using the early fruit fly embryo, Fasulo and colleagues could track many synchronized cell divisions at once, allowing for fast and direct analysis of the effects of the various inhibitors used. The cartoon and images above show the different steps during mitosis of these divisions. Both chromosomes (top row of images, green in merged) and microtubules (middle row of images, red in merged) are fluorescently tagged.

Fasulo, B., Koyama, C., Yu, K., Homola, E., Hsieh, T., Campbell, S., & Sullivan, W. (2012). Chk1 and Wee1 kinases coordinate DNA replication, chromosome condensation, and anaphase entry Molecular Biology of the Cell, 23 (6), 1047-1057 DOI: 10.1091/mbc.E11-10-0832

December 19, 2011

Just when you think the mitotic spindle can’t get any more magical, the Ran pathway comes out and says, “I’m here, beyotch!” Today’s image is from a paper showing how kinetochore fibers are stabilized using a Ran-dependent mechanism.

The mitotic spindle is a complicated apparatus that functions to separate chromosomes during mitosis through the attachment of microtubules to kinetochores on chromosomes. Many of these microtubules are sourced from a pair of centrosomes on either side of the spindle, but there is a population of important microtubules that are not generated at centrosomes. These “acentrosomal” microtubules are instead generated by the presence of RanGTP around the chromosomes. The idea is that these microtubules are able to capture kinetochores easily by being nucleated so close to them. The other ends (minus ends) of these acentrosomal microtubules are focused near the centrosomes, and a recent paper describes how these microtubules are stabilized. A protein called MCRS1 is a RanGTP-regulated protein and is found at the minus ends of chromosomal and kinetochore microtubules, according to Meunier and Vernos. MCRS1 stabilizes kinetochore fiber microtubules, and without it, spindles are unstable. As seen in the images above, MCRS1 (middle row, green in merged) is localized to the minus ends of microtubules (top row, red in merged). MCRS1 localization is more obvious (arrow in higher mag image) when only kinetochore fiber microtubules are present (middle column) when compared with control (left column). When kinetochore fibers are absent (right column), so is MCRS1.

Meunier, S., & Vernos, I. (2011). K-fibre minus ends are stabilized by a RanGTP-dependent mechanism essential for functional spindle assembly Nature Cell Biology, 13 (12), 1406-1414 DOI: 10.1038/ncb2372
Adapted by permission from Macmillan Publishers Ltd, copyright ©2011

September 26, 2011

Our cells ask a lot of our chromosomes. Chromosomes contain all of our genetic material, but are required to compact themselves into skinny little things that can be easily divided during cell division. Throw a terrible virus into the mix, and chromosomes start to have trouble with their duties.

During mitosis, duplicated chromosomes separate after receiving a signal allowing anaphase to begin. Until this signal is relayed, each pair of chromatids stays attached to one another by the cohesin protein complex. Premature chromatid separation was recently found in some types of white blood cells in HIV-infected people, and can lead to cells having an incorrect number of chromosomes. This same research group more recently showed that this HIV-induced premature chromatid separation is caused by Vpr, an HIV accessory protein. Vpr causes premature chromatid separation by disrupting the higher-order structure of DNA surrounding the centromere, the region where kinetochores allow attachment to the mitotic spindle. In the images above, the cohesin complex (pink, arrows) is found within the centromere of a control chromatid pair (left). In cells expressing the HIV accessory protein Vpr (right), the cohesin complex is absent from the centromere of a loosely-bound chromatid pair (arrowheads).

Shimura, M., Toyoda, Y., Iijima, K., Kinomoto, M., Tokunaga, K., Yoda, K., Yanagida, M., Sata, T., & Ishizaka, Y. (2011). Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation originally published in The Journal of Cell Biology, 194 (5), 721-735 DOI: 10.1083/jcb.201010118

July 28, 2011

The centromere is the plate on which a kinetochore is served. That plate, however, is mysterious. Recently, a group of cell biologists dug into this mystery by forming kinetochores on chromosome regions not typically associated with kinetochores.

Kinetochores are multi-protein structures that serve as the sites of spindle attachment for each chromosome during mitosis. Kinetochores are assembled on regions of the chromosomes known as the centromere. Although the importance of the centromere has long been appreciated, the exact qualities of centromere-ness remain unresolved. Some past research shows that centromere location in budding yeast is based on DNA sequence, while other organisms rely on repeated DNA sequences or epigenetic marks (meaning not due to DNA sequence) to identify the location of a centromere. A recent paper describes a technique that triggers brief over-expression of one of those epigenetic marks—the protein CID (Drosophila CENP-A or CENH3 for those keeping score). After a pulse of this over-expression, chromosomes are coated with CID signal (green, middle image above), yet after a few generations of cell division, those CID marks are mostly cleared away (right image). The remaining CID marks (arrows), Olszak and colleagues found, became sites of new functional kinetochores. By monitoring the early steps of kinetochore formation, these biologists hope to understand how centromere position is determined.

Agata M. Olszak, Dominic van Essen, António J. Pereira, Sarah Diehl, Thomas Manke, Helder Maiato, Simona Saccani & Patrick Heun (2011). Nature Cell Biology 13,799–808. DOI: 10.1038/ncb2272.
Adapted by permission from Macmillan Publishers Ltd, copyright 2011

May 12, 2011

Some of my favorite proteins ride on the ends of microtubules. I don’t usually like to anthropomorphize proteins, but I always think of Dr. Strangelove hopping on the growing end of a microtubule (and I didn’t even like that movie!). Today’s image is from a paper that adds a satisfying piece to the mitotic spindle puzzle.

Astrin is a mitotic spindle protein that is required for proper alignment of chromosomes on the metaphase plate during mitosis. It wasn’t clear exactly what astrin is doing to promote chromosome alignment, or how astrin arrives at the right location. A recent paper paints a much more complete astrin story. Dunsch and colleagues found a novel protein called kinastrin that forms a complex with astrin and a few others. Astrin localizes to the plus ends of microtubules, and requires kinastrin to do so. The presence of this complex may be affecting microtubule dynamics directly, consistent with the chromosome alignment and spindle integrity problems seen in kinastrin- and astrin-depleted cells. Image above shows fluorescently-tagged astrin localized to the ends of microtubules. The different colors show the localization of astrin in subsequent time-lapse images (ie, the astrin moves with the end of the microtubule).

Dunsch, A., Linnane, E., Barr, F., & Gruneberg, U. (2011). The astrin-kinastrin/SKAP complex localizes to microtubule plus ends and facilitates chromosome alignment originally published in The Journal of Cell Biology, 192 (6), 959-968 DOI: 10.1083/jcb.201008023

February 7, 2011

Cancer is not a disease…it is many many diseases. Some cancers come about gradually, while others can occur from a single catastrophic cellular event, according to a recent paper.

Most cancers are believed to progress through a series of genetic changes that gradually allows cells to become cancerous and spread. These genetic changes are random, but can be pushed along by carcinogens or DNA repair problems. Sometimes, though, there can be big pulses of genetic change that encourage cancer progression, and a recent paper describes how single catastrophic cellular events can cause major genomic rearrangements that quickly lead to cancer. In this paper, Stephens and colleagues found multiple cancer samples with tens to hundreds of genomic rearrangements that were caused by a single catastrophic event, and termed this phenomenon “chromothripsis.” Evidence of chromothripsis can be found in at least 2-3% of all cancers, including many subtypes, and in about 25% of bone cancers. Images above show chromosomes from a renal cancer cell line, with chromosome 5 marked with different fluorescent probes that find specific genomic regions. Compared to the normal chromosome 5, the derivative chromosome 5 has gross rearrangements suggesting chromothripsis, as seen by the close juxtaposition of all of the fluorescent probes.

Stephens, P., Greenman, C., Fu, B., Yang, F., Bignell, G., Mudie, L., Pleasance, E., Lau, K., Beare, D., & Stebbings, L. (2011). Massive Genomic Rearrangement Acquired in a Single Catastrophic Event during Cancer Development Cell, 144 (1), 27-40 DOI: 10.1016/j.cell.2010.11.055
©2011 Elsevier Ltd. All rights reserved.

September 13, 2010

A telomere is the end region of a chromosome that helps protect it from shortening during DNA replication, and has a repetitive and characteristic DNA sequence. A recent paper looks at the role of a protein called Rap1 in both telomere function and transcriptional regulation. Rap1 is important to prevent telomere recombination (exchange of segments of DNA) and fragility, which can lead to events characteristic of cancer in mammals. Images above show chromosomes from mouse cells without Rap1. The telomere signal on each chromosome strand is labeled with one color, and telomere recombination can be seen when the signals are exchanged (arrows).

Reference: Paula Martinez, Maria Thanasoula, Ana R. Carlos, Gonzalo Gómez-López, Agueda M. Tejera, Stefan Schoeftner, Orlando Dominguez, David G. Pisano, Madalena Tarsounas and Maria A. Blasco. Reprinted by permission from Macmillan Publishers Ltd: Nature Cell Biology 12, 768–780, copyright 2010. Paper can be found here.

August 12, 2010

The centromere is a region on a chromosome where sister chromatids are joined together, and where the kinetochore is assembled in order to attach to the mitotic spindle. Centromeres do not have a specific DNA sequence, but do have several unique features including highly repetitive DNA sequences. A recent paper has found that these repetitive sequences are important to generate a fully functional centromere. The authors used a cell line where one chromosome has relocated its centromere, but left the repetitive sequence at the old centromere’s site. Image above shows the chromosomes from this cell line, with zoomed images of the chromosome with the new centromere. A protein called CENP-A (top) labels the new centromere (arrowhead), while a more general centromere label (bottom) is found on both the new and old (asterisk) centromere sites.

Reference: Emily A. Bassett, Stacey Wood, Kevan J. Salimian, Sandya Ajith, Daniel R. Foltz and Ben E. Black, 2010. Originally published in Journal of Cell Bioloy. doi: 10.1083/jcb.201001035. Paper can be found here.

Great blurb about this paper can be found here.

May 20, 2010

Chromosome architecture is tightly regulated during mitosis, from condensation and cohesion of chromosome arms early in mitosis, to chromosome cleavage and separation in anaphase. A recent paper uncovers a pathway that is critical for proper chromosome architecture; an ATPase called PICH works with the kinase Plk-1 to regulate chromosome arm cohesion and organization during mitosis. Image above shows chromosomes during mitosis, stained for Plk-1 (left, red in merged image), PICH (center, green in merge), and DNA (right, blue in merge).

Reference: Yasuhiro Kurasawa and Li-yuan Yu-Lee. Authors’ Molecular Biology of the Cell paper can be found here.

April 8, 2010

The separation of duplicated chromosomes during mitosis was widely believed to be a random process. However, a group was recently able to distinguish the different DNA strands and regions on the chromosomes, and show the non-random segregation of chromosomes in colon epithelial cells. Image shows mouse chromosomes with these different labels.

Reference: Ester Falconer, Elizabeth A. Chavez, Alexander Henderson, Steven S. S. Poon, Steven McKinney, Lindsay Brown, David G. Huntsman & Peter M. Lansdorp. Adapted by permission from Macmillan Publishers Ltd: Nature 463, 93-97, copyright 2010. Paper can be found here.