The Story Behind the Paper: How calmodulin became efficient

by John Lisman

Story behind: Nat Neurosci. 2011 Mar;14(3):301-4. Calmodulin as a direct detector of Ca2+ signals. Faas GC, Raghavachari S, Lisman JE, Mody I.

Long-term potentiation, a model for memory, is triggered by the activation of the calmodulin-dependent protein, CaMKII in dendritic spines. Sri Raghavachari, my former postdoc, and I were interested in how exactly CaMKII gets activated during LTP. It seemed that it should be straightforward to account for this in a computational model. Our confidence was based on the fact that a lot of groundwork had been done—the elevation of Ca2+ that triggered this process had been measured by Ryohei Yasuda and the interactions of Ca2+, calmodulin and CaMKII had all been determined in test tube experiments. But when Sri put this all together in a standard biochemical model, the simulations indicated that there would be virtually no CaMKII activation. Clearly something was wrong because Ryohei Yasuda had shown that under the same conditions in which he measured Ca2+ elevation in spines, he could also measure strong CaMKII activation.

During the summer I work at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Sri came down to visit. We spent hours trying to figure out what was wrong with the simulations. Sri had carefully checked the simulations and determined that the program could accurately account for other data. Thus, the problem was not a bug in the program, but rather in an assumption we had put into the program. Every day, we awoke, convinced we had found the erroneous assumption; by nightfall we had rejected that idea.

One of the great things about working at MBL is the number of other neuroscientists there and the collegial atmosphere. We went to talk to Bill Ross, an expert in the measurement of Ca2+ in neurons. It had long been known that when Ca2+ enters neurons, very little of it stays free because most gets bound to “buffer” molecules. These are like the pH buffers all biochemists use; it’s just that Ca2+ buffers bind Ca2+ instead of protons. Bill asked us a lot about the particular Ca2+ buffers that were in spines—what was known about their molecular identity and their Ca2+ binding properties. Moreover, he wanted to know what assumptions about the buffers we had built into our simulations.

The answer to this was simple: we had followed the “standard” dogma based on the work of the Nobel Prize winner, Ernst Neher. He had determined that neurons contained a “fast buffer” that very rapidly binds the entering Ca2+. He had not been able to determine what type of molecule this was. Every model of Ca2+ dynamics that had since been developed had incorporated this fast buffer into the scheme and we had followed this convention. Thus, when Ca2+ entered the cytoplasm, 95% got bound to the fast buffer; only the remaining 5% was free and could activate calmodulin.

Perhaps there was something wrong with this assumption, but to evaluate this issue we had to learn about how Ca2+ buffers work, something we knew little about. Fortunately, Isabelle Llano was working as instructor in the MBL Neurobiology course. Because of her expertise in the small proteins that buffer Ca2+ in neurons, we went to chat with her. We learned a lot from her, but she also pointed us to Guido Faas, who was working with Istvan Mody at UCLA and measuring the kinetics of how Ca2+ binds to protein buffers. Previous work had measured the equilibrium properties of Ca2+ binding to proteins—the binding rate was then inferred by calculation. In contrast, Guido was using advanced methods to rapidly jump the Ca2+ concentration and then actually measure how fast Ca2+ would bind.

As we learned more about Ca2+ buffers from Guido and read the literature more carefully about calmodulin, we finally came up with a radical but intellectually satisfying new model—-perhaps the fast buffer that Neher had measured was none other than calmodulin itself. This would certainly radically change our computer simulations—-instead of calmodulin responding to only the 5% of Ca2+ that remained free after the bulk of Ca2+ was soaked up the unknown “fast buffer”, calmodulin would be activated by all the Ca2+ ions that entered, making the process of calmodulin activation and CaMKII activation much more efficient.

But for this to be true, calmodulin would have to bind to Ca2+ fast, faster than to the other major Ca2+ binding proteins in neurons (e.g. calbindin). When we talked to Guido about this possibility, he was excited to test it. His previous work had dealt only with calbindin, but he could now extend the work to calmodulin. Indeed, he could reconstitute the buffering in spines, putting both calmodulin and calbindin into his cuvettes. When the results came in, they were stunning; calmodulin has extraordinarily fast Ca2+ binding kinetics, much faster than that of calbindin.

With the new binding parameters provided by Guido, Sri reformulated his computer model. He took out the unknown fast buffer and replaced it with only calmodulin (which is at surprisingly high concentration) and calbindin. The simulations now showed that enough calmodulin was activated to account for the measured activation of CaMKII, our holy grail.

The new view we propose makes sense: Calmodulin is the transducer that couples Ca2+ entry to enzyme activation. It would make sense for calmodulin to be as efficient a detector of Ca2+ as possible and thus to directly intercept the entered Ca2+. Our results indicate that this is the case. Ca2+ triggered reactions are implicated in hundreds of forms of biological signaling. We therefore believe that this new view of Ca2+ signaling will have broad applicability.

Yeast genetics and familial ALS

In a recent paper in PLoS Biology, “A Yeast Model of FUS/TLS-Dependent Cytotoxicity“, Brandeis postdoc Shulin Ju and coworkers applied yeast genetics to examine the function of the human protein FUS/TLS. The gene for FUS/TLS is mutated in 5-10$ of cases of Familial ALS. The yeast model expressing the mutant protein recapitulates many important features of the pathology.

A particular feature of interest is that  FUS/TLS form cytoplasmic inclusions of this protein which is normally localized to the nucleus. Over-expression of a number of yeast proteins rescues the cells from the toxic effect without removing the inclusions. The results are suggested to implicate RNA processing or RNA quality control in the mechanism of toxicity, which I find really interesting in light of the talk Susan Lindquist (an author on this paper) gave at Brandeis about yeast prions and regulatory proteins earlier this month.

Other authors on the paper include Brandeis professors Dagmar Ringe and Gregory Petsko, and Brandeis alumni Dan Tardiff (PhD, Mol. Cell. Biol.,  ’07), currently a postdoc in the Lindquist lab at the Whitehead Institute,  and Daryl Bosco (PhD, Bioorganic Chem, ’03), currently on the faculty at U. Mass. Medical School.

For more information, please see the paper itself or the longer article about the research on Brandeis NOW.

Detecting Mutations the Easy Way

Recent Brandeis Ph.D graduate, Tracey Seier (Molecular and Cell Biology Program), Professor Sue Lovett, Research Assistant Vincent Sutera, together with former Brandeis undergraduates Noor Toha, Dana Padgett and Gal Zilberberg have developed a set of bacterial strains that can be used as “mutational reporters”.  Students in the Fall 2009 BIOL155a, Project Laboratory in Genetics and Genomics, course also assisted in the development of this resource. This work has recently been published in the journal Genetics.

These Escherichia coli strains carry mutations in the lacZ (β-galactosidase) gene that regain the ability to metabolize lactose by one, and only one, specific type of mutation. This set allows environmental compounds to be screened for effects on a broad set of potential mutations, establishing mutagen status and the mutational specificity in one easy step.

This strain set is improved over previous ones in the inclusion of reporters that are specific for certain types of mutations associated with mutational hotspots in gene. Mutations at these sites occur much more frequently than average and involve DNA strand misalignments at repeated DNA sequences rather than DNA polymerase errors. Such mutations are associated with human diseases, including cancer progression, and have been under-investigated because of the lack of specific assays. Using this strain set, Seier et al. also identified a mutagen, hydroxyurea, used in the treatment of leukemia and sickle cell disease, which affects only the “hotspot” class of mutations. This strain set, which will be deposited in the E. coli Genetic Stock Center,  will facilitate the screening of potential mutagens, environmental conditions or genetic loci for effects on a wide spectrum of mutational events.



Left: E. coli colonies showing lacZ mutant revertants (blue pimples) arising on a white colony on growth medium containing the beta-galactosidase indicator dye,  X-gal


yet more papers in the wild

More papers appearing recently:

Current Brandeis authors noted in boldface, past Brandeis trainees shown in italics

Older Adults are Better at Spotting Fake Smiles

Studies of aging and the ability to recognize others’ emotional states tend to show that older adults are worse than younger adults at recognizing facial expressions of emotion, a pattern that parallels findings on non-social types of perception. Most of the previous research focused on the recognition of negative emotions such as anger and fear. In a study “Recognition of Posed and Spontaneous Dynamic Smiles in Young and Older Adults” recently published in Psychology and Aging, Derek Isaacowitz’s Emotion Laboratory set out to investigate possible aging effects in recognizing positive emotions; specifically, the ability to discriminate between posed or “fake” smiles and genuine smiles. They video-recorded different types of smiles (posed and genuine) from younger adults (mean age = 22) and older adults (mean age = 70). Then we showed those smiles to participants who judged whether the smiles were posed or genuine.

Across two studies, older adults were actually better at discriminating between posed and genuine smiles compared to younger adults. This is one of the only findings in the social perception literature suggesting an age difference favoring older individuals. One plausible reason why older adults may be better at distinguishing posed and spontaneous smiles is due to their greater experience in making these nuanced social judgments across the life span; this may then be a case where life experience can offset the effects of negative age-related change in cognition and perception.

This was the first known study to present younger and older adult videotaped smiles to both younger and older adult participants; using dynamic stimuli provides a more ecologically valid method of assessing social perception than using static pictures of faces. The findings are exciting because they suggest that while older adults may lose some ability to recognize the negative emotions of others, their ability to discriminate posed and genuine positive emotions may remain intact, or even improve.

The Emotion Laboratory is located in the Volen Center at Brandeis. First author Dr. Nora Murphy (now Assistant Professor of Psychology at Loyola Marymount University) conducted the research as a postdoctoral research fellow, under the supervision of Dr. Isaacowitz, and second author Jonathan Lehrfeld (Brandeis class of 2008) completed his Psychology senior honors thesis as part of the project. The research was funded by the National Institute of Aging.

Lights, Camera, Splice!

In their paper “Ordered and Dynamic Assembly of Single Spliceosomes” appearing in Science this week, Brandeis postdoc Aaron Hoskins and co-workers use a combination of yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to work out the kinetic mechanism by which the spliceosome assembles on a model pre-messenger RNA prior to splice out an intron in the RNA. The work is a collaboration between Jefl Gelles’s lab in Biochemistry,  Melissa Moore’s lab at UMass Medical School, and  Virginia Cornish’s lab at Columbia.

Hoskins et al. use a single-molecule fluorescence approach that dubbed “CoSMoS” (Co-localization Single Molecule Spectroscopy), originally developed in the Gelles lab by Larry Friedman and Johnson Chung, that is a powerful method to study the assembly and function of the complex macromolecular machines that perform a wide variety of biological functions. In this movie, shown 150x faster than real time, the comings and goings of many U1 spliceosome components on a surface-tethered pre-mRNA are shown as the appearance and disappearances of white spots.  The white spots orginate from the fluorescence emission of specifically labeled U1 components upon excitation with a 532nm laser.

Pre-mRNAs are spliced in a complex cycle wherein the spliceosome assembles, is activated for catalysis, performs two transesterification reactions, and disassembles on every turnover.  Steps between the isolatable intermediates depicted in this cycle involve the coordinated association and dissociation of many spliceosome components.  A key finding by Hoskins et al. is that spliceosome assembly is reversible, and this is represented by the dashed arrows between the pre-mRNA, A, and B complexes.

The multi-wavelength, total internal reflection fluorescence (TIRF) microscope built by Larry Friedman and Johnson Chung in the Gelles laboratory uses lasers of different wavelengths to excite spectrally distinguishable fluorophores on various spliceosome components. Photo by Diane Katherine Hunt.

According to Hoskins, who will leave Brandeis to take up a faculty position in the Biochemistry Department at the University of Wisconsin, Madison

By far, the most challenging aspect of the project was determining two completely orthogonal methods for attaching fluorophores to endogenous spliceosomes in whole cell extract.  Since these experiments are quantitative, we needed to find methods that give a very high degree of fluorophore incoporation and specificity (in other words, 10% labeling would not cut it!).

The novel part, for me, is that for decades spliceosome kinetics have been “off-limits” to enzymologists due to the complexity of the system.  However, by developing the correct analytical tools, the spliceosome can be studied in detail usually reserved for enzymes orders of magnitude smaller.

Hoskins plans to continue these single molecule studies of the spliceosome in his new lab in Wisconsin and will be focusing on splice site selection and  coupling of nuclear RNA processing events.  He also aims to develop new methodologies for fluorescent labeling of ribonucleoproteins in vitro and in vivo.

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