HighMag: August 2012

August 30, 2012

The time and energy required to understand any complex process in the cell can be intimidating. Frequently biologists will take a systematic approach, whether that is identifying every protein involved, finding the exact location of proteins, or uncovering every protein-protein interaction, etc. Some biologists combine a few approaches, throw in their own clever spin, and turn out a pretty darn complete picture of what’s going on. With that in mind, enjoy today’s image!

Endocytosis is the uptake of material into the cell, and involves a long list of proteins that are recruited to and dissociated from the membrane in a specific order. A recent paper describes how the combination of fluorescent microscopy and electron microscopy was used to systematically correlate the composition of proteins with the plasma membrane’s shape throughout endocytosis in budding yeast. Kukulski and colleagues used fluorescent tags that correlate with different modules, or stages, of endocytosis to understand how the localizations of proteins overlap over time. Then, these fluorescent tags were used as a guide in electron tomography to visualize the exact structure and shape of the membrane at each module. In the images above, yeast cells have fluorescent tags for the coat module, an intermediate step, of endocytosis (green, Sla1) and a later actin module (red, Abp1). When the two proteins overlap at the membrane, the signal appears yellow. Below each fluorescent image is the corresponding electron tomography image that shows the ultrastructure of the membrane at each stage. The cartoon above shows the time window in endocytosis for each protein.

Wanda Kukulski, Martin Schorb, Marko Kaksonen, & John A.G. Briggs (2012). Plasma Membrane Reshaping during Endocytosis Is Revealed by Time-Resolved Electron Tomography Cell, 150 (3), 508-520 DOI: 10.1016/j.cell.2012.05.046

August 23, 2012

Art imitates life, but sometimes it is life that imitates art. And when I say “art,” I’m talking about my dance moves (don’t think it’s “art”?….get your own blog!). So, imagine my happiness at seeing that neutrophils have their own interpretation of my lasso dance move to help them roll within a vessel. Today’s image is from a fascinating paper describing this neutrophil sling.

Neutrophils are white blood cells that are some of the first responders to inflammation. Their migration toward inflamed tissue is made difficult due to the high shear stress of blood flowing through the vessel. Past research showed that neutrophils roll over the vessel’s surface by flattening and using long membrane tethers at the rear of the cell. A recent paper shows that these rear tethers are, in fact, the remnants of a sling that the neutrophil uses to roll within the vessel. Sundd and colleagues show that as a neutrophil rolls forward, a sling wraps itself around the cell and lays in front of the cell. The sling is then used as an adhesive substrate on which the cell can roll easily within the microvessel environment. As the cell rolls forward, the sling is peeled up and the process is repeated. The images above show two different neutrophils using a sling for forward rolling. The back tether of the sling (arrowhead, top images) is swung to the front of the cell (long white arrows, second images down), after which the cell moves forward.

Prithu Sundd,, Edgar Gutierrez,, Ekaterina K. Koltsova, Yoshihiro Kuwano, Satoru Fukuda, Maria K. Pospieszalska, Alex Groisman, & Klaus Ley (2012). ‘Slings’ enable neutrophil rolling at high shear Nature, 488 (7411), 399-403 : doi:10.1038/nature11248

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

August 16, 2012

One of the most fascinating and terrifying things about human health is how a single mutation in a single gene can cause such dramatic disorders and diseases. A person may have a lifetime of adjustments that their disorder or handicap requires, a constant looking-over-your-shoulder for that increased cancer risk (BRCA, I’m looking at you), or far worse. One mutation can affect one small part of a cell that, in no small way, affects everything. Today’s image is from a paper on primary cilia, and discusses applications of the research into understanding human ciliopathies.

Cilia are microtubule-based protrusions that function in sweeping material across a tissue (motile cilia) or as sensory orgnanelles (primary cilia). The link between several disorders and ciliary defects has driven more research towards understanding how cilia are formed and how they function. Specifically, certain disorders that cause blindness result from defective or dying photoreceptor cells in the retina, which have the largest primary cilia found in mammals—rod and cone photoreceptors. In a recent paper, Zhang and colleagues identified the roles of a novel protein, Ttc26, in ciliogenesis. Zebrafish with reduced levels of Ttc26 had ciliary defects both in kidneys and photoreceptor cells. Without Ttc26, cells produced cilia that were short and defective. In the images above of rat photoreceptor cells (green), Ttc26 (red) is seen in the transition zone of cilia. Zhang and colleagues suggest that based on the importance of Ttc26 in ciliogenesis, patients with ciliary disorders should be screened for ttc26 gene mutations.

Zhang Q, Liu Q, Austin C, Drummond I, & Pierce EA (2012). Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish. Molecular biology of the cell, 23 (16), 3069-78 PMID: 22718903

August 13, 2012

Actin does pretty well on its own, but with tropomyosin as wing-man it is a force to be reckoned with. Tropomyosin is necessary for our muscles to contract, and recently was found to be important in vesicle and organelle transport.

Molecular motors such as myosin are frequently described as processive or nonprocessive, or how well they stay on and walk along the actin filaments (or microtubules). The class of myosin V motors is made of unconventional myosins that function in intercellular cargo trafficking. Several class V myosins are nonprocessive, including the budding yeast Myo2p myosin that functions in vesicle and organelle transport. A recent paper describes results on the requirements that make this myosin processive within a yeast cell. Hodges and colleagues found that bare actin filaments cause Myo2p to be nonprocessive, but the addition of tropomyosin, a protein that binds along the length of actin, to the filament allowed the Myo2p to remain strongly attached to actin. In the images above, a schematic shows the attachment of a fluorescent quantum dot to the Myo2p motor (left). The quantum dot (red, yellow arrow) could then be tracked as the motor moved along an actin-tropomyosin filament (green).

Alex R. Hodges, Elena B. Krementsova, Carol S. Bookwalter, Patricia M. Fagnant, Thomas E. Sladewski, & Kathleen M. Trybus (2012). Tropomyosin Is Essential for Processive Movement of a Class V Myosin from Budding Yeast Current Biology, 22 (15), 1410-1416 DOI: 10.1016/j.cub.2012.05.035

August 10, 2012

Biologists are always looking for ways to push the limits of what our microscopes are capable of doing. These Microscopy Olympians (which should be a real thing, seriously) understand the physics and biology behind imaging cells in order to build, tweak, and test fancy-shmancy new technologies. And, just like the real Olympics, I watch from my couch with a bag of pretzels in front of me.

To accurately capture high resolution images of cells, biologists are always improving ways to capture three dimensional structures over time in a living cell. Combining nanoscale resolution with live imaging has proven a challenge, recently helped by a paper that describes the tweaks made to earlier microscopy setups. Paszek and colleagues presented their technique called scanning angle interference microscopy, in which dynamic cellular events and structures are imaged with nanoscale resolution in three dimensions. In the image above, microtubules imaged using this technique show their location in three dimensions. The “height” along each microtubule is color-coded (in nm) to show that microtubules bend down towards the cell cortex.

Paszek MJ, Dufort CC, Rubashkin MG, Davidson MW, Thorn KS, Liphardt JT, & Weaver VM (2012). Scanning angle interference microscopy reveals cell dynamics at the nanoscale. Nature methods, 9 (8), 825-7 PMID: 22751201

August 6, 2012

Morphogenesis may be a big word, but the concept is simple and beautiful. Morphogenesis refers to how cells become shaped and organized into complete tissue or organs….and eventually an actual organism. Morphogenesis is what countless developmental biologists think about as they fall asleep, as they wake up, as they shower…how about that for obsession?! Today’s image is from a paper on epithelial morphogenesis—specifically the formation of tube structures in a liver.

Understanding how cells rearrange themselves into tissue structures is a main goal of developmental biology. Many structures rely on epithelial cells that line a lumen. For example, the tubular bile ducts of the liver are made of epithelial cells that both regulate the composition of bile and prevent leakage of bile along the tube. These epithelial cells are called cholangiocytes, and a recent paper paves the way towards understanding how these cells form the tube structures during development. In this paper, Senga and colleagues identified a transcription factor called grainyhead-like 2 (Grhl2) that regulates the size of a lumen surrounded by epithelial cells during development. Grhl2 upregulates claudin 3 and claudin 4, components of the tight junctions that provide a tight barrier between epithelial cells. Grhl2 also targets Rab25, which in turn increases claudin 4 levels and regulates its localization at tight junctions. As seen in the images above, expression of Grhl2 (bottom row) in cysts of liver progenitor cells causes rapid expansion of the lumen inside of the cysts without increasing the number of cells, compared with the slow formation of the lumen in a control cyst (top row).

Kazunori Senga, Keith E. Mostov, Toshihiro Mitaka, Atsushi Miyajima, & Naoki Tanimizu (2012). Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25 Molecular Biology of the Cell, 23 (15), 2845-2855 DOI: 10.1091/mbc.E12-02-0097

August 2, 2012

Sometimes you watch a movie, and then you watch it again. And again. And again. Maybe it was Goonies, back when you were a kid (or adult)…or it was a pair of otters holding hands or a baby panda sneezing. Or maaaaybe it was watching stem cells and their progeny divide in real time, within the actual organism. Thanks to the technical gymnastics of a group of stem cell researchers, we can now see how stem cells and their progeny within a hair follicle divide and regenerate hair.

Tissue regeneration begins with stem cells and their immediate progeny. Knowledge of this process has been incomplete due to the difficulty of tracking all of the cell behaviors in living tissue. A group recently used high resolution imaging to track hair regeneration within hair follicles of mice over a long period of time. By tracking cell behaviors, Rompolas and colleagues found that the immediate progeny of stem cells actively divide to regenerate the tissue, while stem cells divide more slowly to contribute to hair regeneration over time. The stem cell progeny cell divisions are mostly aligned with the long axis of the hair. Finally, Rompolas and colleagues used cell ablation to show that a mesenchymal group of cells called the dermal papilla is required for hair regeneration. In the images above, stem cell progeny divisions can be seen in a live hair follicle undergoing growth. Three different cells are colored to show their divisions (right columns).

BONUS! Check out a movie of stem cell progeny divisions here and here, and a movie of stem cell divisions here. For all movies from this paper, click here.

Panteleimon Rompolas, Elizabeth R. Deschene, Giovanni Zito, David G. Gonzalez, Ichiko Saotome, Ann M. Haberman, & Valentina Greco (2012). Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration Nature, 487 (7408), 496-499 DOI: 10.1038/nature11218
Adapted by permission from Macmillan Publishers Ltd, copyright ©2012