September 25, 2014

While taking an awesome cell biology course in college, I was coming to terms with my mother’s recent ovarian cancer diagnosis. The scientist in my head couldn’t shake the curiosity about how my mother’s cells could have betrayed her so royally. This intersection of basic cell biology and cancer kick-started my interest in cell biology research. Today’s image is from a paper showing a role for the ARF tumor suppressor in maintaining chromosomal stability. THIS paper is one of the million billion reasons why basic research is necessary and important. 

The ARF tumor suppressor is mutated or absent in many cancers, and is known to stabilize p53 in response to cellular stress. Other, p53-independent roles for ARF contribute to its role as a tumor suppressor, but are not well understood. A recent paper describes ARF’s function in chromosome segregation during mitosis, via Aurora B regulation. Britigan and colleagues show that loss of ARF results in aneuploidy, or an incorrect number of chromosomes, stemming from chromosome segregation and spindle organization defects. These defects can be rescued through overexpression of the Aurora B kinase, which helps ensure proper kinetochore-spindle interactions and is overexpressed in some cancers. In the images above, ARF-/- cells (right column) show defects throughout mitosis, when compared to normal cells (left). Defects include misaligned chromosomes during metaphase (top, middle rows), and lagging chromosomes during anaphase (bottom).

Britigan, E., Wan, J., Zasadil, L., Ryan, S., & Weaver, B. (2014). The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation of Aurora B Molecular Biology of the Cell, 25 (18), 2761-2773 DOI: 10.1091/mbc.E14-05-0966

June 10, 2014

The Life History of a Single Kinetochore Fiber sounds like a book a lot of us would enjoy (well, me at least). It isn’t really a book about a plucky kinetochore fiber who triumphs over a difficult childhood, but rather the focus of a fascinating recent paper. In this paper published in Molecular Biology of the Cell, LaFountain and Oldenbourg present results showing a model for kinetochore microtubule formation that occurs at kinetochores.

Kinetochore fibers link chromosomes to the mitotic spindle, which drives chromosome segregation during anaphase. The prevailing model of kinetochore fiber formation includes a “search and capture” mechanism, in which some dynamic spindle microtubules reach a kinetochore and become stabilized by the interaction. A recent paper by LaFountain and Oldenbourg shows, however, that the maturation of these kinetochore fibers depends on the addition of microtubules at the kinetochore-proximal end, with polymerization towards the spindle pole. In this study, the naturally birefringent microtubules of crane-fly spermatocytes were examined, allowing a quantitative analysis of where microtubules are added. In the images above, kinetochore-proximal addition of microtubules can be seen in the centrosome-free half-spindle (red arrows) of a crane-fly spermatocyte, from early prometaphase to metaphase (top to bottom).

LaFountain, J., & Oldenbourg, R. (2014). Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes Molecular Biology of the Cell, 25 (9), 1437-1445 DOI: 10.1091/mbc.E14-01-0008

April 2, 2014

Nuclear envelope breakdown is far prettier than my own breakdown when I realized that Girl Scout “cookie season” is over. Today’s image is from a paper that describes the importance of SUN proteins in nuclear envelope breakdown.

Early in mitosis, a cell’s nuclear envelope breaks down to allow the attachment of chromosomes to the mitotic spindle. Nuclear envelope breakdown (NEBD) depends on a tearing process, during which microtubules pull the nuclear envelope towards the centrosomes. Turgay and colleagues found that the SUN proteins help to clear membranes from chromatin during NEBD. SUN proteins reside in the inner nuclear membrane and are part of a complex that connects the nucleus to the cytoskeleton. As seen in the images above, simultaneous depletion of both SUN1 and SUN2 (bottom timelapse) delayed removal of the nuclear envelope (green; chromosomes in red), when compared to control (top).  

Turgay, Y., Champion, L., Balazs, C., Held, M., Toso, A., Gerlich, D., Meraldi, P., & Kutay, U. (2014). SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown The Journal of Cell Biology, 204 (7), 1099-1109 DOI: 10.1083/jcb.201310116

April 16, 2013

I’m as type-A as a person can get, with my organized desk, to-do lists, and inability to roll with it (I sound dreadful, right?).  One thing that will never change is the calm I feel when I read the word “systematic” in a paper’s title or abstract.  Anything done systematically soothes me like a heartbeat soothes a newborn.  Today’s image is from a paper that uses a—you guessed it—systematic approach to understanding the roles of dynein and its many regulators in mitosis.

Dynein is a large microtubule motor complex that is important in countless cellular processes, most notably mitosis.  Like most proteins, dynein relies on numerous adaptor proteins and the dynactin complex to help localize the motor and/or activate it.  A recent paper uses siRNA screening to systematically test the roles of dynein subunits, adaptor proteins, and dynactin subunits to build a more complete picture of the roles of each protein in mitosis.  Raaijmakers and colleagues show that while some regulators are required for activation but not localization of dynein, others are required mainly for dynein localization.  Dynactin, for example, is not necessary for spindle organization, but rather serves as a dynein recruitment factor at the nuclear envelope and kinetochores.  In the images above, the mitotic spindle in a control cell is very focused at spindle poles (second row, green in merged).  When the dynein heavy chain subunit is depleted from cells (all other columns), spindles show a range of spindle pole focusing defects, including spindles lacking attachment to their poles.  Spindle microtubules are top row, red in merged; chromosomes are third row, blue in merged.

Raaijmakers, J., Tanenbaum, M., & Medema, R. (2013). Systematic dissection of dynein regulators in mitosis originally published in the Journal of Cell Biology, 201 (2), 201-215 DOI: 10.1083/jcb.201208098

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.

March 22, 2013

If there was an NCAA-type bracket of cool things to image in a cell, I’m pretty sure a spindle would make it pretty far in the tournament—if not take the whole thing.  They are extremely photogenic, and their important role and dynamic nature make them a top seed for sure.  Today’s image is from a paper identifying a new player in spindle positioning.

The position of the mitotic spindle serves as the guide to where the cell will be divided into two daughter cells in cytokinesis.  The spindle is positioned with the help of astral microtubules—microtubules that grow from the centrosomes toward the cell periphery.  The interaction of these microtubules with machinery at the cell’s cortex can generate pulling or pushing forces that position the entire mitotic spindle, yet this interaction isn’t completely understood.  A recent paper identifies a protein called MISP as a player in the microtubule-cortex interaction.  Zhu and colleagues found that depletion of MISP, an actin-binding protein, resulted in shortened astral microtubules and rocking, misoriented spindles, among other mitotic defects.  Zhu and colleagues showed that MISP is phosphorylated by Plk1, a major kinase important in many mitotic processes.  The images above show MISP (gray, red in merged images) localization throughout mitosis.  MISP is localized to cortical actin (green).  Boxed regions are shown in higher magnification in the insets.  

Zhu M, Settele F, Kotak S, Sanchez-Pulido L, Ehret L, Ponting CP, Gönczy P, & Hoffmann I (2013). MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression. originally published in the Journal of Cell Biology, 200 (6), 773-87 PMID: 23509069

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

September 24, 2012

If you are a mitotic spindle aficionado or superfan, then you understand how the mitotic spindle has a life of its own. Information on the spindle’s composition, dynamics, and list of duties can fill entire volumes…and there is still so much we are learning about the spindle. Today’s image is from a paper that adds another chapter in our metaphorical mitotic spindle book.

The mitotic spindle is composed of dynamic microtubules and countless proteins that ensure chromosomes are equally segregated into identical daughter cells at the end of cell division. Because much of the structure of the spindle comes from the cytoplasm, the contributions from the nucleus have remained unclear. A recent paper identifies the long-elusive presence of a nuclear matrix—a viscous matrix of proteins that supports the formation and function of the spindle. The nuclear matrix is composed of nuclear proteins that reassemble after nuclear envelope breakdown into a pole-to-pole structure. By looking at the cell divisions in fruit flies, Yao and colleagues found that the nuclear matrix is distinct from spindle microtubules, and is found closely around chromosomes following drug treatments that depolymerized microtubules. The time-lapse images above show mitosis in the syncytial Drosophila embryo. The nuclear matrix (green, Chromator) has a spindle-like morphology as the microtubule spindle (red, tubulin) itself forms. 

Yao C, Rath U, Maiato H, Sharp D, Girton J, Johansen KM, & Johansen J (2012). A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis. Molecular biology of the cell, 23 (18), 3532-41 PMID: 22855526

August 20, 2012

When you watch a cell going through mitosis, it looks like a smooth ballet performance—grace with impeccable timing and synchrony. In reality, there is a lot going on within the cell to make mitosis progress so smoothly, just like the ballet dancers’ actual physical exertion and concentration. Today’s image is from a paper that describes the timely removal of proteins from the nuclear envelope during mitosis.

The nuclear envelope (NE) is a double membrane that separates the cell’s genome from the rest of the cell, and permits transport of material into and out of the nucleus through multi-protein complexes called nuclear pores. During mitosis, the NE breaks down in order to allow chromosomes to attach to the mitotic spindle. Prior to NE breakdown, the nucleoporins that make up the nuclear pore complexes must be dissociated from the NE. A recent paper describes the timely removal of the nucleoporin NPP-3 near centrosomes at the onset of mitosis in early worm embryos. Hachet and colleagues found that centrosomes and the Aurora-A kinase AIR-1 are both required for removal of NPP-3 from the NE. In the images above, NPP-3 (right column, red in merged) is localized on the NE and is removed as mitosis progresses. NPP-3 removal begins near centrosomes (microtubules in green). 

Hachet V, Busso C, Toya M, Sugimoto A, Askjaer P, & Gönczy P (2012). The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos. Molecular biology of the cell, 23 (16), 3111-21 PMID: 22740626

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

March 5, 2012

Scenario: You’re at my house for a dinner and I make you a mind-blowing chocolate tart. You ask why it is so durn good, and I pass along the fact that Nutella is the ass-kicking ingredient. Next thing you know, you’re trying to add Nutella to everything at home….and by golly, it makes (many) things taste better. Today’s image is from a paper characterizing the relationship between the many cellular changes after treatment by antimitotic drugs. Knowing how these drugs work (by analogy, finding the ass-kicking “ingredient”) can help folks develop improved anti-cancer drugs.

Many anticancer drugs are antimitotic drugs, meaning they function by blocking the progress of cell division during mitosis. In addition to arresting mitosis, these drugs cause apoptosis, DNA damage, and induction of p53 (a tumor suppressor gene), and a recent paper investigates the relationship between all of these events. After treating cells with powerful antimitotic drugs, Orth and colleagues found that the resulting prolonged mitotic arrest (or slippage from that arrest) causes DNA damage, which in turn causes an induction of p53. The DNA damage was inhibited when these treated cells were prevented from launching the pathway for apoptosis, which is programmed cell death. So, Orth and colleagues concluded that the prolonged mitotic arrest caused by antimitotic drugs results in a partial activation of apoptosis. Understanding this partial apoptotic response in the context of treating tumors should help guide development of improved cancer therapies. The images above show increasing DNA damage (red spots) after prolonged treatment with an antimitotic drug, compared with an untreated cell (top left). By 16 and 48 hours of drug treatment (bottom), cells had very high levels of DNA damage. Arrows point to mitotic cells.

Orth, J., Loewer, A., Lahav, G., & Mitchison, T. (2011). Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction Molecular Biology of the Cell, 23 (4), 567-576 DOI: 10.1091/mbc.E11-09-0781

January 9, 2012

As a reader of this blog, I bet you are simply dazzled by mitosis like I am. Or, you may be sick of mitosis and my love of mitosis images. If that’s case, then scram! Mitosis is one of the most photogenic events in a cell and today’s images support this widely-accepted claim.

During mitosis, a lot has to happen correctly for two daughter cells to have an equal number of chromosomes. The list of participating proteins is as long as my arm, and includes several kinases that regulate mitotic progression. Aurora B kinase participates in just about every major mitotic event—it regulates chromosome condensation, localizes to microtubules, functions in monitoring and ensuring chromosome attachment to the spindle, and is necessary for cytokinesis. The role of a similar kinase, Aurora A, is less clear, possibly due to differences in the techniques used in previous research that led to ambiguous or contradictory results. Hégarat and colleagues recently used a chemical genetic strategy to find that Aurora A kinase is important in chromosome alignment and segregation. In addition, Aurora A and Aurora B kinases cooperate together to coordinate chromosome segregation and microtubule dynamics. Images above show different mitotic cells after Aurora A kinase depletion (chromosomes are blue, spindle is green, spindle poles are red). Many of the cells appear normal, and may represent different stages of mitosis. Some cells displayed gross defects in spindle morphology, as seen as the presence of multipolar and monopolar spindles (bottom right two images).

Hegarat, N., Smith, E., Nayak, G., Takeda, S., Eyers, P., & Hochegger, H. (2011). Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics originally published in The Journal of Cell Biology, 195 (7), 1103-1113 DOI: 10.1083/jcb.201105058

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

November 28, 2011

Asymmetric cell division results in two unequal daughter cells and without it, stem cells would not hold the attention of biologists, sick patients, politicians, and those loud nut-jobs who have more passion than knowledge. Check out today’s image, from a paper identifying a new protein complex important in asymmetric division.

Asymmetric division occurs throughout development, and is when a cell divides to result in two daughter cells that are unequal in size and/or cell fate. Part of this process is the establishment of the axis along which the cell will divide. Once this polarity is established, the mitotic spindle can line up accordingly. Fruit fly neural stem cells, called neuroblasts, divide asymmetrically to result in the birth of another neuroblast and a smaller ganglion mother cell, which eventually gives rise to neurons. A recent paper identifies a new role for a signaling complex in establishing neuroblast polarity. In this paper, Carmena and colleagues found that the Rap1–Rgl–Ral complex of proteins regulates polarity establishment in fruit fly neuroblasts, and works alongside other well-studied polarity proteins such as aPKC, Par6, and Pins. As seen in the images above, Rap1 (bottom row, green in merged; arrows) is enriched on only one side (apical side) of neuroblasts throughout division (DNA is in red).

Carmena, A., Makarova, A., & Speicher, S. (2011). The Rap1-Rgl-Ral signaling network regulates neuroblast cortical polarity and spindle orientation originally published in The Journal of Cell Biology, 195 (4), 553-562 DOI: 10.1083/jcb.201108112

October 24, 2011

Breaking up is hard to do. Thankfully for us, breaking up is also very beautiful (in cells). Abscission is the final cleaving of two daughter cells at the end of mitosis, and is really quite stunning to see. So, enjoy today’s images!

Cytokinesis is the physical division of two daughter cells at the end of mitosis. The final step of cytokinesis is abscission, during which the small midbody that connects the two cells is finally cleaved. This process involves precise regulation of cytokinesis proteins; for example, the small GTPase RhoA is required during cytokinesis for the establishment and contraction of the cleavage furrow that develops to divide the cells, yet RhoA must be inactivated for abscission. A kinase protein called CIT-K (citron kinase) was previously shown to function as a downstream effector of RhoA activity, yet a recent paper describes results suggesting the converse—that CIT-K regulates RhoA activity. In addition, Gai and colleagues found that CIT-K also interacts with and regulates anillin, an actin scaffold protein crucial in cytokinesis. The images of midbodies above show the localization of either anillin (left, green) or RhoA (right, green), as well as DNA (blue) and microtubules (red). Compared with control cells (top row), anillin and RhoA were nearly undetectable at late stage midbodies in cells lacking CIT-K (bottom row).

Gai, M., Camera, P., Dema, A., Bianchi, F., Berto, G., Scarpa, E., Germena, G., & Di Cunto, F. (2011). Citron kinase controls abscission through RhoA and anillin Molecular Biology of the Cell, 22 (20), 3768-3778 DOI: 10.1091/mbc.E10-12-0952

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

September 15, 2011

There is something so gratifying about a light switch. My two-year old will pull a chair to our kitchen light switch to turn it on and off. Over. And over. And over again. Maybe that’s why I find phosphorylation so satisfying (and maybe why I have a headache). It’s a molecular switch, and the vast combinations of where, when, and how different proteins are phosphorylated can provide mind-numbing levels of regulation within a cell. Combine my appreciation for phosphorylation with my absolute love for early worm embryos, and you have today’s lovely images.

The one-cell stage worm embryo divides like many cells throughout development—asymmetrically. Asymmetric cell division results in two daughter cells with different developmental fates and frequently different sizes. For asymmetric cell division to take place in the early worm embryo, the entire mitotic spindle apparatus is moved towards one end of the cell, the posterior. A complex of polarity proteins (made of PAR proteins and the aPKC homolog PKC-3) functions upstream of an evolutionary conserved pathway of proteins (made of the NuMA homolog LIN-5 and G-protein signaling), and a recent paper finds the well sought-after link between these two pathways. In Galli and colleagues’ paper, they show that LIN-5 is phosphorylated by PKC-3. The position of PKC-3 at only one side of the cell results in the phosphorylation of LIN-5 only in that region, which in turns allows the mitotic spindle to position itself correctly. In the images above, one-cell stage worm embryos show staining for phosphorylated LIN-5 (top row, red in bottom row) during mitosis (spindle is in green, chromosomes in blue). Phosphorylated LIN-5 is enriched at higher levels at the anterior cortex (the left-hand side in each image) during earlier stages of mitosis.

Galli, M., Muñoz, J., Portegijs, V., Boxem, M., Grill, S., Heck, A., & van den Heuvel, S. (2011). aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division Nature Cell Biology, 13 (9), 1132-1138 DOI: 10.1038/ncb2315
Adapted by permission from Macmillan Publishers Ltd, copyright ©2011

September 8, 2011

So after lab meeting, I’m going to grab a drink at the plus end of a microtubule. I hear it’s a swingin’ place, with lots of cool things going on. I also hear a kinesin-8 is a real mover and shaker at the plus end, but you’ll have to check out today’s image to see for yourself.

The structure and function of the mitotic spindle depends on the amazing qualities of microtubules. Microtubules grow and shorten from their plus ends (mostly), which are the ends that reach out towards the periphery of the cell or chromosomes during mitosis. The plus ends of microtubules are hotspots of activity—plus-tip-tracking proteins bind to the plus ends of microtubules and can regulate the dynamics of each microtubule individually. A group recently found that a microtubule motor called KIF18B plays an important role in regulating microtubule dynamics at the plus end. According to Stout and colleagues, KIF18B controls the length of astral microtubules, which are those that extend towards the cell periphery, and does so by interacting with another well-studied plus-tip-tracking protein called EB1. Without KIF18B, cells have an increased number and length of microtubules. As seen in the images above, KIF18B (green) is on the ends of astral microtubules during different stages of mitosis, and EB1 (orange) is found on the ends of all microtubules. Microtubules are gray, and chromosomes are blue.

Stout, J., Yount, A., Powers, J., LeBlanc, C., Ems-McClung, S., & Walczak, C. (2011). Kif18B interacts with EB1 and controls astral microtubule length during mitosis Molecular Biology of the Cell, 22 (17), 3070-3080 DOI: 10.1091/mbc.E11-04-0363

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

May 5, 2011

So much depends on healthy cell division, so it is no wonder how magnificent the spindle checkpoint is. This checkpoint keeps our cells healthy and our biologists busy as they try to figure it all out.

The spindle checkpoint ensures that a dividing cell undergoes anaphase only when all chromosomes are properly attached the mitotic spindle. Without this checkpoint, cells may end up with an incorrect number of chromosomes. Recently, a group teased apart some of the specifics of the checkpoint by using a construct engineered in the lab. This construct fused the kinetochore protein Mis12 to the checkpoint protein Mad1, which blocks progression to anaphase when it localizes to kinetochores not attached to the spindle. This Mad1-Mis12 construct targeted Mad1 to kinetochores despite their orientation state on the mitotic spindle, meaning that this construct allowed researchers to distinguish between Mad1 checkpoint signaling and the initial orientation error signal. The images above show metaphase spindles in control cells (top) or cells with the Mad1-Mis12 construct (bottom). The construct (mCherry, red in merged image) localizes to kinetochores (CREST, blue in merged) attached to the spindle (tubulin, green).

Maldonado, M., & Kapoor, T. (2011). Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation Nature Cell Biology, 13 (4), 475-482 DOI: 10.1038/ncb2223
Adapted by permission from Macmillan Publishers Ltd, copyright 2011