Mary Hoff

DNA Amplification and Detection Made Simple (Relatively)

Twenty-three years ago, a man musing about work while driving down a California highway revolutionized molecular biology when he envisioned a technique to make large numbers of copies of a piece of DNA rapidly and accurately. Known as the polymerase chain reaction, or PCR, Kary Mulliss technique involves separating the double strands of a DNA fragment into single-strand templates by heating it, attaching primers that initiate the copying process, using DNA polymerase to make a copy of each strand from free nucleotides floating around in the reaction mixture, detaching the primers, then repeating the cycle using the new and old strands as templates. Since its discovery in 1983, PCR has made possible a number of procedures we now take for granted, such as DNA fingerprinting of crime scenes, paternity testing, and DNA-based diagnosis of hereditary and infectious diseases.

Inhibiting Hedgehog: New Insights into a Developmentally Important Signaling Pathway

What transforms a ball of undifferentiated cells into an organism with a nervous system, digestive tract, and other specialized body parts? Among the proteins that play an important role is one with the unlikely name of hedgehog (Hh). When Hh attaches to a transmembrane protein known as patched (Ptch1), it initiates a series of molecular interactions that lead to activation of the transcription factor Gli (for “glioma associated”) and the onset of key events in embryonic differentiation. We know this process involves freeing a second transmembrane protein, smoothened (Smo), from inhibition. But how do Hh and Ptch1 accomplish this? Scientists would like to know because the ability of this signaling pathway to function properly makes the difference between normal development and devastating abnormalities—and because the pathway is also implicated in tumor growth. Maarten F. Bijlsma, Maikel P. Peppelenbosch, and colleagues began their attempt to find out by noting that previous studies show that enzymes used to make cholesterol are involved in the pathway; that Ptch1 and Smo don’t necessarily bond to each other; that Ptch1 looks like other proteins that pump molecules from one side of the cell membrane to the other; and that cholesterol-like molecules can inhibit the pathway. Based on that information, the researchers hypothesized that when—and only when—Ptch1 is unencumbered by Hh, it pumps a cholesterol-like molecule into the extracellular space, where it inhibits Smo. To test this, the researchers developed an experimental system made up of fibroblasts modified to luminesce when Gli is active (called reporter cells), and to overexpress various combinations of Ptch1, Smo, and Gli. When they mixed reporter cells overexpressing Smo with cells overexpressing Ptch1, Gli activation in the reporter cells was reduced. When they mixed them instead with cells in which Ptch1-producing genes were silenced, Gli activation increased. After performing additional tests to eliminate alternative explanations, the team concluded that Ptch1 inhibits Smo through an intermediary, and that the intermediary molecule can exert its influence between individual cells. The researchers next exposed reporter cells to a medium that had contained Ptch1-overexpressing cells, and found Gli activation to be strongly inhibited. However, when they exposed reporter cells to a serum-free, Ptch-conditioned medium, they found no inhibition. Since serum-free medium doesn’t contain lipoproteins, they concluded that a lipoprotein is involved. Further tests suggested that the lipoprotein acts by helping transport a 3β-hydroxysteroid involved in the pathway inhibition. Interestingly, the researchers noted that certain people with Hh signaling problems have elevated levels of a particular hydroxysteroid, 7-dehydrocholesterol (7-DHC). This led them to test the link between 7-DHC and Gli activity in mouse cells, which in turn led to the conclusion that 7-DHC indeed participates in Ptch1’s inhibition of Smo. But is the Smo-inhibiting molecule actually 7-DHC or a compound derived from 7-DHC? When the researchers exposed medium from 7-DHC-producing mouse cells to ultraviolet radiation—which changes 7-DHC into vitamin D3—or used vitamin D3 in place of 7-DHC, Hh pathway inhibition was even stronger. They also showed that vitamin D3 binds to Smo, and that it inhibits the Hh pathway in live zebrafish embryos. Putting it all together, the researchers concluded that when Hh isn’t present, Ptch1 pumps (pro)-vitamin D3 (i.e., either 7-DHC or vitamin D3) into the extracellular space, where the hydroxysteroid grabs onto Smo, inhibiting Gli activation. When Hh binds to Ptch1, the pump grinds to a halt, Smo is freed of inhibition, and transcription of developmentally important genes kicks in. This new knowledge of the Hh signaling pathway shows how some cells can affect neighboring cells’ development, and it helps explain some of the problems associated with mutations affecting cholesterol biosynthesis. Because the Hh pathway is linked with certain cancers, it also has implications for tumor development. Additional work is now underway to see whether this new understanding of Ptch1’s intercellular inhibition of Smo can be applied to help suppress tumor growth.

Surviving salt: how do extremophiles do it?

​ImmersedImmersed in waters saltier than chicken soup, salt-tolerant “halophilic” microorganisms are able to thrive in conditions that would reduce a less-adapted organism to a shriveled remnant. One way halophilic archaea avoid this fate is by bathing their molecular machinery in a similarly salty intracellular environment that would cause ordinary proteins to lose their shape. How do the proteins inside these cells survive?

Loopy Chromatin Brings Distant DNA to Bear on Silencing Promoter Genes

What makes an unspecialized cell become heart or skin or brain or tumor? The answer—or at least part of it—lies in the presence of proteins that attach to DNA and act like switches to turn on transcription in genes that are involved in guiding that cell toward one destiny or another. The presence of such proteins, in turn, depends on an elaborate system for silencing and activating the genes that make them. Scientists are only beginning to understand this system, which they hope to manipulate to use stem cells for therapeutic purposes and to stop the development of cancer cells. In a new study, Stephen Baylin, Vijay Tiwari, and colleagues shed light on this gene regulation system by exploring the silencing and activation of a gene, Gata-4, that is implicated in switching on genes that promote cell differentiation and—when abnormally suppressed—in promoting tumor development. The researchers focused on Polycomb group (PcG) proteins, which attach to large stretches of DNA in early embryonic cells to help keep key developmental regulatory genes at a low level of expression, but disappear when the cells begin to differentiate. Studies in fruit flies and mammals suggest that PcG proteins are involved in regulating the transcription of these regulatory genes. But its not clear how PcG proteins—particularly those not near the genes—exert their influence. Its also not clear how PcGs interact with DNA methylation—the attachment of methyl groups, like bits of bling, to DNA that is known to be involved in keeping genes turned off. To learn more about PcGs role in regulatory gene silencing and activation, Baylin, Tiwari, and colleagues tapped a variety of biochemical tools to look at the molecular makeup of chromatin in the vicinity of Gata-4 under various circumstances in four kinds of cells: undifferentiated embryonic carcinoma cells (Tera-2), in which Gata-4 is expressed at a low rate; Tera-2 cells that had been induced to differentiate and in which Gata-4 is expressed at very high levels; adult colon cancer cells, in which Gata-4 is fully silenced and the DNA near Gata-4 is hypermethylated; and adult colon cancer cells that were genetically altered so that DNA was not methylated around Gata-4 and Gata-4 is re-expressed, but at a low level. The “loopiness” of chromatin around a target gene influences the genes level of transcriptional activation. Using two techniques designed to determine how various chromatin (the DNA–protein network that makes up chromosomes) regions physically situate with respect to each other within the chromosome—associated chromosome trap (ACT) assay and chromosome conformation capture (3C) analysis—the researchers found that distant portions of chromatin interacted with Gata-4 in Tera-2 cells, creating chromatin loops that clustered around it. With the chromatin in this configuration, Gata-4 was expressed at a low level. A similar analysis on Tera-2 cells that had been induced to differentiate showed that the interactions had disappeared and the chromatin was almost linear with respect to the original loops, suggesting that the loops were related to transcription potential. Applying the same tests to adult colon cancer cells, the researchers found that chromatin was super-loopy in the hypermethylated, super-silenced version. In the cells that had been genetically altered to eliminate methylation, however, Gata-4 was expressed at a low rate, and loops were present but less abundant—a state reminiscent of the undifferentiated Tera-2 cells. These results indicate that loops and gene silencing are related. To further elucidate the role of PcG proteins, the researchers looked for various forms of PcG related to another kind of methylated area (called histone marks) along the chromatin on either side of Gata-4. They found that the PcGs occurred far upstream and downstream of the gene in undifferentiated Tera-2, but disappeared in the cells that had begun to differentiate. The adult colon cancer cells showed levels of PcG and associated marks at the Gata-4 gene that were lower than those in the Tera-2 cells but higher than those found in very active genes in the adult cancer cells. Together, these findings suggest that the PcGs and histone marks might be helping to silence the gene through the looping process. To test this, the researchers applied RNA interference to deplete PcGs in Tera-2 cells. They found that loopiness decreased and transcription increased slightly— strong evidence that PcGs play a role in the looping. Similarly, in the silenced adult colon cancer cells, but not in the ones expressing Gata-4, methylated bits of DNA occur a long distance from the gene and are brought close to the gene by looping, providing additional evidence that chromatin loopiness works to silence Gata-4. The researchers concluded that chromatin looping mediates a “poised” (low expression) state by bringing transcription-blocking PcG and associated histone marks close to the gene in embryonic cells, and that a similar state, when associated with DNA hypermethylation, leads to super-silencing in adult colon cancer cells. The researchers note that their findings have important implications for cancer treatment and prevention. They show that gene silencing is more complicated than previously thought based on a linear rather than looped chromatin configuration. Thus, the cause and effect of loopiness will need to be taken into consideration in future efforts to explore the modification of silencing of key genes such as Gata-4 as a tool for preventing or treating cancer.

An Unexpected Connection: Potassium Limitation and Ammonium Toxicity in Yeast

Give the yeast Saccharomyces cerevisiae the right growing conditions and it multiplies like crazy—as any bread maker or beer brewer can testify. But deprive it of sufficient potassium, and it’s lucky to survive. Why? Since S. cerevisiae is a model organism for eukaryotes, the answer to that question could provide valuable insights into cellular processes of many organisms, including humans. To learn how potassium limits growth in yeast, David C. Hess, David Botstein, and colleagues enlisted the assistance of DNA microarray analysis, a biochemical tool that allows scientists to identify which genes are active in a cell at any given time. What they found gave them a start: in S. cerevisiae cells grown in a potassium-limited medium, genes involved in a seemingly unrelated process—nitrogen metabolism—showed dramatically altered activity compared with unstressed cells, with genes repressed by products of nitrogen-compound breakdown becoming less active, and those whose products facilitate amino acid transport showing increased activity. At first, that made about as much sense as discovering that every time you open your refrigerator the stock market drops. What could possibly be the connection? The altered gene activity pattern suggested an attempt to deal with a toxic influx of nitrogen within the cell, but nitrogen toxicity has been thought to be limited to multicellular organisms, with one-celled types easily able to keep the nutrient in balance by excreting excess through cell membrane channels. Could limited potassium upset that ability? In search of an answer, the scientists looked at cells exposed to different ammonium and potassium levels. They found that in low-potassium but not high-potassium environments, cell numbers went down dramatically as ammonium concentration increased, suggesting that ammonium is indeed toxic to yeast when potassium is limited. A second test, in which they increased concentration of the nitrogen-rich amino acid asparagine rather than ammonium, confirmed that what they were seeing was not a general nitrogen effect, but one specific to ammonium. Further tests of other strains of S. cerevisiae confirmed that they were not dealing with a situation unique to a single quirky cell type. If what they were seeing was indeed an adverse reaction to ammonium, the researchers predicted they should also see some sort of metabolic fingerprint of the yeast’s efforts to detoxify its environment. And they did. In collaboration with the Rabinowitz lab at Princeton, they used liquid chromatography tandem mass spectrometry to test the biochemical contents of medium in which ammonium-stressed yeast cells were grown. There, the researchers found high levels of amino acids—apparently the yeast equivalent of the urea we mammals excrete in urine to remove toxic nitrogen from our system. Having confirmed the presence of ammonium toxicity, the researchers next turned their attention to the issue of the mysterious connection with potassium concentration. Because potassium and nitrogen have similar chemical properties, they hypothesized that ammonium ions leak into cells through potassium channels when those channels are not otherwise occupied ushering potassium across the cell membrane. To test this, they engineered strains of S. cerevisiae in which ammonium influx into the cells could be increased without stimulating innate ammonium concentration regulatory mechanisms. Even in high-potassium environments, cells engineered to let in lots of ammonium showed greater mortality than those engineered to let in little, supporting the hypothesis that excess influx of ammonium is the root of the problem. Furthermore, the researchers found that in engineered cells in which ammonium transport across the cell membrane was high, growth was indeed limited even though potassium was not, and the cells excreted high levels of amino acid, mimicking the potassium-limited state. The researchers concluded that S. cerevisiae does indeed experience ammonium toxicity under potassium-deprived conditions and that it uses a primitive detoxification system involving the production and excretion of amino acids in an attempt to deal with it. On a broader level, they demonstrated that systems biology techniques such as microarray analysis and mass spectrometry are valuable resources for discovering and exploring biochemical relationships and pathways that might otherwise remain masked in the normal workings of healthy cells. They hope in further studies to use these and other emerging tools to learn whether similar ammonium toxicity is also found in bacteria and to elucidate the mechanism behind S. cerevisiae’s amino acid–based detoxification system. For more on ammonium toxicity, see the related Primer (DOI: 10.1371/journal.pbio.0040388).

Ping-pong positioning: alternating protein interactions at actin filament barbed ends helps establish polarity in mammalian oocytes.

​ForFor mammalian egg cells to form successfully, the precursor cell (the oocyte) must divide asymmetrically, forming a large egg that contains the storage material required for embryo development, and a small polar body that receives surplus chromosomes. How does the oocyte manage this asymmetrical division? Key to the answer is a meshwork of actin filaments that moves the chromosome-segregating spindle, initially formed at the center of the oocyte, toward the cell cortex. Mouse oocyte, showing the spindle undergoing translocation toward the cortex. Actin network - cyan, chromosomes - magenta. The meshwork-building process involves three actin-binding proteins named profilin, Formin 2, and Spire (also known to display genetic interactions in Drosophila), which cooperate in vivo. But exactly how do these three proteins work together to accomplish this important task? A new in vitro study by Marie-France Carlier, Pierre Montaville, and colleagues has revealed a fascinating ping-pong like interaction between Formin 2 and Spire acting on profilin-bound actin. It was known that the C-terminus of the FH2 domain of Formin 2 associates with the N-terminal (Nt) KIND domain of Spire. The present team previously found that when Nt-Spire alone (composed of the KIND domain and 4 WH2 domains) associates with actin barbed ends via its WH2 domains, it blocks their growth from profilin-actin. On the other hand, Formin 2 (like other formins), is expected to promote processive filament barbed end assembly from profilin-actin. Thus Spire and Formin 2 individually have antagonistic effects on actin assembly. How then can synergistic actin assembly arise from the two proteins together? The authors here use a combination of bulk solution and single filament (microfluidics-assisted TIRF microscopy) polymerization assays to reconstitute synergistic actin assembly in vitro and provide mechanistic insight. They show that the FH1-FH2 domain of Formin 2 by itself poorly nucleates filaments from profilin-actin and Nt-Spire alone inhibits assembly, but together they display enhanced assembly. Barbed end growth assays establish that the two proteins interact together at filament barbed ends to promote this synergy, and that their direct mutual interaction is mediated by association of the KIND domain of Nt-Spire with the FH2 C-terminal region of Formin 2. However by itself, the KIND domain inhibits filament assembly. Thus the WH2 domains of Spire are involved in the synergistic behavior. The team also demonstrates that in vitro, at steady state conditions mimicking the in vivo context, the amount of assembled actin is determined by the relative amounts of Spire and Formin 2. The exact molecular mechanism responsible for the puzzling synergistic effect of Spire and Formin 2 is established by single filament assays. The team shows that while individual filaments grow slowly from profilin-actin in the absence of Spire or Formin 2, they switch to arrested growth when the barbed ends are capped by Spire. Formin 2 binds unusually slowly to barbed ends, but when it does it promotes very fast processive assembly. Remarkably, Formin 2 binds 30-fold faster to Spire-capped ends than to free barbed ends, where it is recruited by Spires KIND domain. Spire actually saddles Formin 2 at the barbed ends, promoting fast processive assembly, which accounts for the synergistic assembly. Spire then rapidly dissociates from the barbed end. Conversely, association of Spire to a Formin 2-bound barbed end arrests fast growth. When Spire and Formin 2 are present together in solution, the filaments display alternating phases of fast and arrested growth, corresponding to alternating Formin 2-bound and Spire-bound states, each protein kicking the other off via a transient state in which both are bound to the filament end. The exact kick-off mechanism remains unknown but may involve ATP hydrolysis on actin. Indeed, filament depolymerization assays show that Spire and Formin 2 bind together cooperatively at depolymerizing ADP-bound barbed ends and block disassembly. Finally, the team looked at the system in vivo by injecting oocytes with Nt-Spire, the KIND domain of Spire, or the FH1-FH2 domains of Formin 2. They found that Nt-Spire alone or FH1-FH2 alone caused an increase in actin filament growth, while KIND alone had the opposite effect and FH2 depressed filament assembly. These data are consistent with and validate the relevance of in vitro studies. But how does this facilitate asymmetric division? The answer to that has to do with the recently observed presence of Formin 2 and Spire on Rab11a positive vesicles in oocytes, and the associated myosinVb-promoted vesicle movement toward the oocyte cortex. The authors tentatively propose that Rab11a-vesicles constantly initiate new filaments via co-association of Spire and Formin 2 to barbed ends. At the same time, the other end of filaments disassembles, creating a pool of profilin-actin fueling filament assembly. The plasticity and myosinVb-enhanced dynamics of this treadmilling network facilitate the slow displacement of the spindle toward the cortex, breaking the symmetry that ultimately leads to egg development. Montaville P, Jegou A, Pernier J, Compper C, Guichard B, et al. (2014) Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism. doi:10.1371/journal.pbio.1001795

A real stretch: mechanisms behind cell elongation.

Does tightening your belt make you taller? You might be tempted to conclude so after learning the results of an intriguing study of how notochord cells elongate in embryos of a primitive sea creature. Interaction of various members of the actomyosin network is essential for the elongation of single notochord cells during the development of Ciona intestinalis embryos. Embryonic development involves two basic processes: cell multiplication and cell shape changes. Elongation in particular is a crucial process for development of the notochord, which serves as a “backbone” for the developing animal and sets the stage for the organization of the rest of the body. It is also particularly intriguing: Elongation begins with the formation of a constrictive actomyosin ring around the middle of the coin-shaped cell, transforming it into an hourglass-shape and eventually into an elongated, drum-shaped cell. Is actomyosins belt-tightening action responsible for notochord elongation? If so, how does it turn squeeze into stretch? To shed light on the process, Di Jiang, Ivonne Sehring, Bo Dong, and colleagues took a close look at the formation, components, and activity of the actomyosin network that appears at the midsection of elongating notochord cells in the sea squirt Ciona intestinalis, which serves as a model chordate for development biology. In other cells, the actomyosin ring is best known for being an integral part of the mechanism that splits one cell into two in the process of cell division, or cytokinesis. Along with actin and myosin, two proteins that work together in a ratchet-like formation to create force, the ring features a number of other proteins that help run the show, including actin-depolarizing factor/cofilin (which severs actin), tropomyosin (which appears to be involved in regulating the stability of actin), α-actinin (an actin regulator), and talin (which helps the actomyosin connect with the plasma membrane during cytokinesis). The researchers first looked to confirm that the presence of the actomyosin ring was not due to cell division activity by looking for evidence of cell cycle processes, such as DNA replication. Finding none, they concluded that the equatorial constriction was not part of a cryptic cell cycle but was instead part of the cell elongation process. Next, the team turned to elucidating the structure, formation, and function of the actomyosin ring. Using immunohistochemistry and fluorescent fusion protein analyses, they discovered that, as in the case of the actomyosin ring present in cell division, the area in which the constriction occurs is rich in cofilin, tropomyosin, α-actinin, and talin — all regulators of actomyosin ring contraction. Using time-lapse photography and other methods, they showed that cortical flow within the cytoplasm was responsible for the recruitment of both actin and myosin to the ring formation site. By creating mutants lacking various components of the ring infrastructure, they discovered that properly functioning cofilin and α-actinin are both needed for cell elongation. In other words, the structure and function of the notochord-lengthening actomyosin ring strongly resembles the ring responsible for cytokinesis, even though its job (lengthening the cell vs. splitting it in two) is very different. Time-lapse photography of elongating notochord cells show frequent membrane deformations occurring at the basal surface during the elongation process, with a two-part cycle creating bleb-like formations that then retracted. Wondering what the deformations connection might be to the constriction of the cells midsection, the researchers looked at the molecular composition of the bleb. They discovered the presence of tropomyosin, cofilin, and the myosin-activating factor MRLC, suggesting that the retraction of the bleb-like deformation is due to individual contractions at the surface and not connected to the compression at the midsection of the cell. Further analysis revealed that as the bleb forms, the apical membrane moves toward the middle of the cell, then returns—suggesting that forces involved in basal cortex bleb retraction help generate the force needed to stretch the cell, contributing to the overall lengthening process. Interestingly, the cells at either end of the notochord do not develop a waist or elongate. To determine whether that is a characteristic of the cell or of the location (having only one notochord cell neighbor), the researchers cut off the ends of the notochord. The new “end cell” also lacked the constriction and elongation function, confirming that having two notochord cell neighbors is key to the elongation process. Putting it all together, the researchers concluded that notochord elongation in the model chordate they studied is due to a combination of equatorial constriction by an actomyosin ring that is essentially identical to the one that transforms one cell into two during cytokinesis, combined with local basal actin–driven contractions in the cytoplasm that help transform cellular belt-tightening into cellular stretch. Finally, intrigued by the idea that an actomyosin complex known primarily for its cytokinetic function is also responsible for an entirely different job in non-dividing notochord cells, the researchers looked to other species in search of a more generalizable way of looking at the systems biological function. Close to home, they found a similar notochord-lengthening function in another tunicate, Oikopleura dioica. In addition, a literature search also revealed that actin-myosin complexes have been implicated in contraction functions in a diverse number of other species and organs, including fish eyes, amphibian nerves, plant roots, and fruit fly ovaries. The ubiquity of the structure and function, the authors concluded, suggests that such constriction is a widely used biophysical solution for lengthening biological units, from cells to entire embryos. Sehring IM, Dong B, Denker E, Bhattachan P, Deng W, et al. (2014) An Equatorial Contractile Mechanism Drives Cell Elongation but not Cell Division. doi:10.1371/journal.pbio.1001781

The Ticket to Transport

Cell nuclei are like gated communities—quite selective about who gets in. And understandably so, because if the wrong proteins showed up at the wrong time and place, the consequences could be disastrous. The standard procedure for moving large molecules that cannot diffuse from the cytoplasm into the nucleus is to use the transport proteins known as karyopherins as escorts. How do karyopherins know whether their cargo is a protein that ought to get in? By their ability to bind with recognition sites on the cargo: no recognition, no passage. The best known example is the canonical or classical nuclear localization signal (cNLS)—a specific sequence that, when added to a protein, drives its nuclear localization. But more recently, a new class of NLSs has been identified, known as a proline–tyrosine nuclear localization signal (PY-NLS). This new signal is recognized specifically by the highly conserved transporter molecule Karyopherinβ2. Bioinformatics studies suggest that there’s a lot of wiggle room in the sequence of the PY-NLSs—a valuable trait, since a variety of molecules need transporting into the nucleus. What are the common features that give them all the ability to connect with Karyopherinβ2? Can these common features be characterized in a way that makes it possible to identify other proteins containing PY-NLSs? In a new study, Yuh Min Chook, Katherine Suel, and Hongmei Gu investigated these questions by focusing on Karyopherinβ2, abbreviated as Kapβ2 in humans and Kap104p in yeast. With Kap104p as a model system, the researchers used mutation and thermodynamic analyses to identify the properties shared by PY-NLSs that successfully bind Kapβ2. The researchers began by confirming that Kap104p substrates do indeed contain PY-NLSs. Then they began to explore the interaction between several human and yeast PY-NLSs and Kap104p. They found that Kap104p recognizes a subclass of PY-NLSs that is characterized by the presence of basic residues among its amino acid constituents, and that human PY-NLSs have an additional hydrophobic subclass that Kap104p doesn’t recognize. They also compared Kapβ2 and Kap104p and learned that about half of the places where Kapβ2 bound with PY-NLSs were also found on Kap104p. This information allowed them to predict whether Karyopherinβ2 from other species would recognize the hydrophobic subclass of PY-NLS. Three linear epitopes (basic region, R, and PY) comprise the PY-NLS. Green fluorescent protein (GFP)-NLS of the nuclear protein Nab2p, which has a PL sequence motif, is visualized in yeast cells. To find out which regions within the NLSs influenced their ability to enter the nucleus, the researchers methodically replaced amino acids at the binding regions of two PY-NLSs. This approach allowed them to identify not only which sections were important for gaining entry to the nucleus, but also which could accommodate variations without inhibiting transportability. The researchers also determined the energy required for various altered cargo molecules to bind their transport molecule, Kap104p, and compared the results to the thermodynamics of four previously characterized PY-NLSs. From this analysis, the researchers reached four conclusions. First, each PY-NLS has three regions that link up to the carrier: one near the N terminal of the NLS, and two closer to the opposite end. Second, the amino acids can vary substantially within each region. Third, the regions are fairly independent from each other in terms of the strength of their connection to the carrier. And fourth, the relative binding affinity of the three regions varies from one PY-NLS to another: mutations in some regions in some PY-NLSs substantially affected binding, while others had little if any effect on the ability to bind Karyopherinβ2. With that knowledge in hand, the researchers assessed the effect of PY-NLS mutations on nuclear uptake in live yeast using two different fluorescence-tagged human proteins containing PY-NLSs. They found that mutations that decrease PY-NLS binding affinity to Kap104p in the test tube also compromise translocation in living yeast cells. The researchers noted that the amount of “give” in binding affinity for this transport system provides latitude for alterations in the amino acid composition of the PY-NLSs without causing them to lose their functionality—a trait that opens the door to supporting the evolution of new functions without sacrificing old ones. Indeed, the researchers point out, some PY-NLSs have been shown to have other jobs, underscoring the adaptive nature of the built-in flexibility. The enhanced understanding of the PY-NLS/Karyopherinβ2 interaction is valuable because it helps establish the parameters for future genome searches for new Karyopherinβ2 substrates. More broadly, it provides important insights into the complex relationships between transport proteins and the substances they carry, which will help guide research into other carrier–cargo interactions and other processes involving recognition of proteins by characteristics that are only weakly related to their sequence.

Picking the Right Parts at the Beta Barrel Factory

Tube-shaped protein constructs known as beta barrels are used by Gram-negative bacteria as passageways for molecules that need to travel from one side of the outer cell membrane to the other. Getting beta barrels embedded into the membrane is a daunting task because of the nonpolar nature of the membrane’s insides; the way cells manage that is with the assistance of a tubular factory already embedded in the membrane that takes in and assembles the outer membrane proteins (OMPs) that comprise the beta barrel. A protein called Omp85 is a central component of this factory in a surprising number of bacterial species as well as in mitochondria and chloroplasts (eukaryotic organelles that arose from bacteria).

Heads, tails, and tools: morphogenesis of a giant single-celled organism.

It’s (relatively) easy to conceptualize how multicellular organisms, like plants and people, generate the specialized structures they need for everyday life: cells differentiate to take on different tasks, such as photosynthesizing and breathing. But what about one-celled organisms? How do their components segregate within a single cell to develop head-to-tail polarity and unique structures, such as cilia? In search of an answer, Mark M. Slabodnick, Wallace F. Marshall, and colleagues turned to Stentor coeruleus, a single-celled aquatic filter-feeder that has remarkably distinct body parts, including cilia, an oral apparatus it uses to ingest bacteria, and a foot-like holdfast with which it clings to surfaces. This giant ciliate—it measures close to a millimeter from head to toe—is renowned for its ability to regenerate from just a tiny part of itself. In fact, because its size allowed elegant surgical manipulations, Stentor became a classical model organism for studying shape and pattern formation in single-celled organisms in the first half of the 20th century. But somehow Stentor fell off the cell biologist’s map in the 1970s: it was never developed as a molecular or genetic model system, and work on the organism largely stopped at that time. Slabodnick, Marshall, and colleagues now put Stentor back on the map by providing the first molecular analysis of regeneration in this organism. These researchers began by testing whether RNA interference (RNAi) could be used to study Stentor morphogenesis. RNAi is a common experimental tool that exploits natural regulatory pathways, allowing the silencing of specific gene transcripts in order to discern how the proteins they encode work. Normally these pathways use small endogenous RNA molecules called microRNAs to target the silencing activity to specific transcripts, but by introducing artificial RNA molecules (siRNAs), scientists can hijack the microRNA system for their own purposes. A look at portions of Stentor’s genome revealed to the authors that it contains genes that code for proteins needed for the microRNA pathway (such as Argonaute and Dicer proteins). To see if this machinery could function to silence Stentor’s gene transcripts, as it does in other organisms, the researchers fed to Stentor bacteria that produce double-stranded RNA molecules that can give rise to siRNAs targeting a gene that encodes a protein, a-tubulin, involved with shaping cellular structures. If the RNAi process worked as expected, they should see as a result the loss of the protein. Indeed, after several days, the bacteria-fed Stentor lost their linear form and became globular. Further analysis using antibodies against a-tubulin confirmed that RNAi had successfully caused the loss of this protein, resulting in the abnormal development of Stentor by disrupting the tubulin-mediated organization of the macronucleus and of cell structures called the cortical rows. Having confirmed that RNAi works in Stentor, the research team then used this approach to study this organism’s regeneration and shape development. Knowing from others’ work that the regeneration of Stentor’s oral apparatus after injury is similar to oral apparatus generation during cell division, the team homed in on the protein Mob1, which is known to play a role in morphogenesis in multicellular organisms, as a likely candidate for regulating shape development in Stentor. Using an antibody to Mob1, they learned that the enzyme concentrates in Stentor’s tail as well as in the region around the oral apparatus. To determine whether and how Mob1 is involved in shaping Stentor, the team then made bacteria containing RNAi vectors targeting Mob1 and fed them to Stentor. Cells that received the RNAispiked bacteria lost their wineglass shape, then either became elongated and curved or developed a blob-like shape with multiple posteriors and oral apparatus regeneration sites. Curious to learn more about the formation of these shapes, the researchers photographed RNAi recipients every two hours after treatment. They found that oral apparatus regeneration was followed by development of a new posterior in the wrong place, and concluded that Mob1 is needed for oral apparatus and posterior structure localization. They also observed that when Mob1 was depleted by RNAi, it was first lost at the mouth end in elongated cells, and later entirely absent as seen in the blob-like cells—suggesting that Mob1 does different jobs in different places.