Beth Brainerd at Histochemistry 2012
Beth takes the audience on a tour of striated muscle anatomy, then introduces the mystery she hopes QSTORM can help her solve – How do muscles contract? By what specific molecular mechanism? We see preliminary confocal and QSTORM images comparing the absorption of quantum dots and Alexa dyes in zebrafish and rabbit psoas models, after microinjection.
Karine and Carol Lynn teamed up to write this summary of the Research Update Presentation delivered by Beth Brainerd at the Athens, Georgia Meeting on December 11, 2011.
BETH BRAINERD’S LAB: Muscle Research
Beth’s QSTORM group at Brown includes graduate student Cally Harper and undergraduate students Julia Olszewski and Natividad Chen. For QSTORM, this group of evolutionary biologists are developing QD labeling techniques for striated muscle cells, and providing one of the project’s two case studies in biological research. They want to better understand how muscles contract, and, at a cellular level, the mechanisms by which titin, actin and myosin proteins coordinate and carry out the work of muscle contraction.
Background: An orientation to the world of striated muscle
Beth began her presentation with a helpful orientation to the confusing Russian doll-like microworld of muscle cell anatomy. (AMAZING FACTOID – ONE HUMAN MUSCLE CELL CAN BE AS LONG AS HALF A METER!).
Beth further outlined the biological research questions she is hoping to address through QSTORM. These include the following:
- Is there any meaningful variation in vertebrates, from species to species, in the lengths of their myosin thick filaments? If so, what governs that variation? (So far, it seems that all vertebrates have 1.6 micron long thick filaments, while the thick filament lengths in invertebrates vary widely.)
- What is the mechanism by which the titin protein contributes to muscle contraction? (It is known that titin binds to actin in the presence of calcium; but, is it just serving as a scaffold, or could it be a winding filament, as one recent hypothesis suggests?) Titin is an especially intriguing protein; it has the highest molecular weight of any molecule and the longest RNA transcription time, suggesting great complexity.
Microinjection and Labeling
Beth’s students started by mastering microinjection techniques, getting a tiny splurt of fluorescent dye into one single muscle cell without destroying it. They could check their handiwork through the lens of a confocal microscope, and practice until they were pros at it.
Microinjecting Jessica’s Quantum Dots
For reasons as yet unexplained – but greatly speculated upon in this meeting – Jessica’s micelle-coated QDs kept passing through the membranes of the single cells into which they were injected, and spread – either to other adjacent cells or into an extracellular matrix. (Are the MQDs destroying the cell membranes? Could there have been a contaminant on the pipette?) Ge commented that his students had experienced the same frustration. Further tests are needed.
Immunohistochemistry with MQDs?
Not only have Jessica’s micelle-coated QDots mischievously infiltrated through cell membranes, but Beth is as yet unsure how to attach to them the antibodies which will be necessary for targeting them to specific muscle proteins within the cells. Jessica said she will prepare them in advance with biotin or carboxyl groups to which antibodies may be attached.
Targeting Dyes to Specific Structures
Not yet able to control the movement of the QDs, and not yet able to attach targeting molecules to them, the team moved on to practice targeting using just fluorescent dye, in vitro. They attached “primary” antibodies to distinct molecules in muscle (actin, myosin, titin, collagen) and “secondary” antibodies to the dye molecules. The two types of antibodies attract and bind to each other. In this case, they were used to bring green dye to collagen and red dye to the three active muscle proteins, yielding a lovely whole embryo image. Eventually they hope to be able to use the antibodies to bind Jessica’s QDs to the protein structures they want to study in living fish. In their wildest dreams they see three different color-emitting QDs distinguishing actin, myosin, and titin simultaneously while the muscle fiber contracts. There’s still a long way to go.
The Fish Fly to Georgia
After getting the best close-ups they could at Brown with their confocal microscope, Beth’s group sent the red and green dye-delineated fish to Peter’s lab for imaging. [This kicked off the notorious “Where is the fish?” episode chronicled in the Team Blog in July 2011.] That’s when Peter’s group first confronted the reality of dealing with “thick” biological samples. The results were less than satisfying.
I’ll Have the Filet…
Peter was still eager to look at some biology using the STORM technique, and so Beth’s gang sent down some thin, fixed (i.e. dead) sections of dye-labeled mouse tongue and rabbit psoas muscle. The psoas is the “filet mignon” muscle, the hip flexor, and its tenderness is due to its relative lack of collagen. This characteristic also makes it easy to tease apart individual muscle fibers for examination. These sections are thin enough for Peter to image. Beth’s students labeled the titin in green and the myosin heads in red, and got these images with their confocal microscope.
However, initial STORM imaging efforts of the psoas sections were not too successful – Andrew reported that the mounting media caused the Alexa dye to bleach too quickly. So, Beth’s team will try a different mounting medium. They want to stick with the rabbit psoas for a while until they get the antibody labeling working. This will also give Peter’s team time to gear up the technique of using induced astigmatism for imaging thicker samples. One very cool feature about the psoas muscle is that even though it has been fixed and glycinerated (not living), it will still contract when treated with an ATP and calcium solution, offering the possibility of imaging the muscle proteins in action.
- Julia’s request to Peter, Andrew and Ben: Please use a scale bar in your images!
- Beth’s team will try a different mounting media for psoas muscle, to avoid dye photobleaching, and retry STORM imaging of Alexa 488 labeled myosin and titin.
- Develop a secondary antibody QD labeling and targeting technique for psoas muscle proteins.
- Develop dynamic muscle contraction methods with QD labels.
- Continue development of techniques for measuring myofilament lengths in vertebrates.
- Check out reports of unusually short thick filaments in jaw depressor muscles of frogs.