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

May 21, 2013

“LET THERE BE LIGHT!” said the microscopist.  Light plays a crucial role in microscopy and cell biology, and a recent paper describes the use of light to understand protein secretion.

Light is used in microscopy in countless ways—to illuminate a sample, excite a fluorophore, and signal the localization or dynamics of a protein.  Light can also be used to manipulate cellular events through the use of “caged” compounds that become active after illumination by certain wavelengths of light.  This technology gives biologists the ability to spatially and temporally control cellular events in order to understand them better.  Recent advances in this technology use illumination of plant photoreceptors to control protein-protein interactions, but some cellular processes such as protein secretion have been difficult to manipulate.  A recent paper describes the use of the plant photoreceptor UVR8 in the first light-triggered protein secretion system developed.  Chen and colleagues have shown that the recently described UVR8 can conditionally sequester proteins bound for secretion in the ER, and then upon illumination with UV light releases these proteins to the plasma membrane.  In the images above, a neuron before (left) and after (right) UV illumination with this UVR8 system shows the movement of proteins known to be secreted from the soma and dendritic processes (arrowheads), where the ER is distributed, and into the Golgi (arrow), a necessary step in protein secretion.

Chen, D., Gibson, E., & Kennedy, M. (2013). A light-triggered protein secretion system originally published in the Journal of Cell Biology, 201 (4), 631-640 DOI: 10.1083/jcb.201210119

May 13, 2013

Molecular motors are some of the raddest things in a cell.  They can walk along cytoskeletal elements such as microtubules and actin filaments, and the list of cellular events that they participate in is a long, long list.  Today’s image is from a paper showing a beautiful pattern of nonmuscle myosin II in epithelial cells.

Epithelial cells assemble junctions to adhere to one another, and the actin motor nonmuscle myosin II (NMII) is a major component of these epithelial apical junctions.  NMII helps the epithelial sheet respond to morphogenesis and changes in tissue homeostasis, and a recent paper describes how the network of NMII motors does so.  Ebrahim and colleagues have found that NMII in the apical junctional complex of epithelial cells assembles into precise muscle-like sarcomere units that form a belt around each cell.  The sarcomeres of neighboring cells are aligned, in turn assembling into a contractile network that can result in changes in cell shape.  In the images above, NMII (green) is seen in repeated sarcomere units around each cell (actin is in red).  NMII puncta are paired together in neighboring cells.  Arrows (middle) point to the junctions between three cells, seen at higher magnification on the right.

Ebrahim, S., Fujita, T., Millis, B., Kozin, E., Ma, X., Kawamoto, S., Baird, M., Davidson, M., Yonemura, S., Hisa, Y., Conti, M., Adelstein, R., Sakaguchi, H., & Kachar, B. (2013). NMII Forms a Contractile Transcellular Sarcomeric Network to Regulate Apical Cell Junctions and Tissue Geometry Current Biology, 23 (8), 731-736 DOI: 10.1016/j.cub.2013.03.039 
Copyright ©2013 Elsevier Ltd. All rights reserved.

May 9, 2013

When we think of wounds, we don’t typically think of them as part of normal, healthy function.  Micro-wounds, however, form when white blood cells have to cross the barrier in our blood vessels to get to an injury or infection.  These micro-wounds happen all the time, and our cells heal these wounds efficiently and elegantly.

One of the most important barriers in our body is that created by the vascular endothelium.  Vascular endothelial cells line all of our blood vessels—from the largest vessels to the smallest capillaries—and function in fluid filtration, hormone trafficking, and recruitment and trafficking of blood and stem cells.  The movement of cells, for example white blood cells, across the vascular endothelium and out of circulation creates “micro-wounds” that can compromise the integrity of the tissue.  A recent paper describes how these micro-wounds are healed, based on a model in which the vascular endothelium senses a loss of tension upon micro-wounding and triggers its own repair.  Martinelli and colleagues tracked micro-wounds that were created by either transmigrating white blood cells or by mechanical disruption by a probe, and found that ventral lamellipodia are generated by endothelial cells to close the micro-wounds.  These lamellipodia are enriched in Rac1 effector proteins, and require reactive oxygen species (ROS) and Arp2/3 for efficient wound closing.  Images above show probe-induced wounding of an endothelial cell, followed by wound healing.  The wound initially expanded to 20um across (80 seconds), with multiple nodes of ventral lamellipodia (blue arrowheads) and ventral F-actin waves forming around the wound and closing it.

Martinelli, R., Kamei, M., Sage, P., Massol, R., Varghese, L., Sciuto, T., Toporsian, M., Dvorak, A., Kirchhausen, T., Springer, T., & Carman, C. (2013). Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds originally published in the Journal of Cell Biology, 201 (3), 449-465 DOI: 10.1083/jcb.201209077

May 6, 2013

Our bodies have multiple fronts for battling viruses, and it’s impressive that any of those suckers manage to invade our bodies at all.  When virus particles do make their way into a cell, it’s important for biologists to understand their pathway through a cell in order to create drug therapies and vaccines.  Today’s image is from a paper describing the use of high resolution imaging to understand this process.

The polarized cells that line our digestive and respiratory tracts form a tight barrier that protects our bodies from viruses.  Understanding how viruses are able to breach these polarized epithelial cells is important in guiding the development of therapeutics and vaccines, yet previous research has focused mainly on in vitro studies of virus entry into nonpolarized cells.  Microscopy advances have recently allowed the high-resolution imaging of virus entry in polarized epithelial cells.  Boulant and colleagues used live-cell spinning-disk confocal microscopy to follow the uptake of single mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs) in polarized Madin–Darby canine kidney cells.  Both virus particles were internalized by clathrin-mediated endocytosis at the apical surface.  MRV virions reached early and late endosomes, while ISVPs escaped the endocytic pathway prior to reaching early endosomes.  In the images above, the tight-junction protein ZO-1 (red, left) shows the typical belt pattern surrounding the polarized cells (side views are also shown in the top and side strips).  The MRV cell surface receptor JAM-A (red, right) is localized near tight junctions and on the apical side of the cells.

Boulant, S., Stanifer, M., Kural, C., Cureton, D., Massol, R., Nibert, M., & Kirchhausen, T. (2013). Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin-Darby canine kidney cells Molecular Biology of the Cell, 24 (8), 1196-1207 DOI: 10.1091/mbc.E12-12-0852

May 2, 2013

If cells had their own soundtracks, I think any flagella-wielding cells would take home the prize.  Maybe the soundtrack begins on a high note with Devo’s “Whip It!”, continues on to the more crass Clarence Carter, plateaus with some headbanging death metal, and finally pays homage to Flock of Seagulls simply due to their flagellar waveform hair.  Today’s image is from a paper describing an outer-inner dynein link in flagella.

Flagella are whip-like organelles protruding from cells, and function to move fluid past the cell to generate motility.  In flagella, nine doublets of microtubules are bundled around a central microtubule pair (the 9+2 structure).  Outer dynein arms (ODAs) and inner dynein arms (IDAs) drive movement of the microtubule doublets past each other, generating the flagellar beating motion.  The ODA and IDA play distinct roles in flagellar function, but a recent paper finds a link between them.  Oda and colleagues found that intermediate chain 2 (IC2) of ODAs functions as part of the outer-inner dynein linker.  IC2 is a hub between ODAs and IDAs to regulate flagellar beating, based on the beating motion of IC2 mutants in the green algae Chlamydomonas.  The images above show the waveforms and image sequences of swimming Chlamydomonas cells.  The bending of flagella in wild-type cells (top row) is different from that of ic2 mutant cells (bottom row).  The principle bend at the flagellar tip in the forward stroke persists in the mutant (arrowhead); this altered bending results in slower swimming for the mutants.

Oda, T., Yagi, T., Yanagisawa, H., & Kikkawa, M. (2013). Identification of the Outer-Inner Dynein Linker as a Hub Controller for Axonemal Dynein Activities Current Biology, 23 (8), 656-664 DOI: 10.1016/j.cub.2013.03.028
Copyright ©2013 Elsevier Ltd. All rights reserved.