The CLIP-170 N-terminal domain binds directly to both F-actin and microtubules in a mutually exclusive manner.

The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. However, a full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein CLIP-170 has been implicated in this actin-MT coordination by associating with the actin-binding signaling protein IQGAP1 and by promoting actin polymerization through binding with formins. Thus far, the interactions of CLIP-170 with actin were assumed to be indirect. Here, we demonstrate using high-speed cosedimentation assays that CLIP-170 can bind to filamentous actin (F-actin) directly. We found that the affinity of this binding is relatively weak but strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170-F-actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170-F-actin and CLIP-170-MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170-F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for MT end-binding protein 1.

Cytoskeletal diversification across 1 billion years: What red algae can teach us about the cytoskeleton, and vice versa.

The cytoskeleton has a central role in eukaryotic biology, enabling cells to organize internally, polarize, and translocate. Studying cytoskeletal machinery across the tree of life can identify common elements, illuminate fundamental mechanisms, and provide insight into processes specific to less-characterized organisms. Red algae represent an ancient lineage that is diverse, ecologically significant, and biomedically relevant. Recent genomic analysis shows that red algae have a surprising paucity of cytoskeletal elements, particularly molecular motors. Here, we review the genomic and cell biological evidence and propose testable models of how red algal cells might perform processes including cell motility, cytokinesis, intracellular transport, and secretion, given their reduced cytoskeletons. In addition to enhancing understanding of red algae and lineages that evolved from red algal endosymbioses (e.g., apicomplexan parasites), these ideas may also provide insight into cytoskeletal processes in animal cells.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

MyTH4-FERM myosins have an ancient and conserved role in filopod formation.

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.

Evolutionary cell biology: two origins, one objective.

All aspects of biological diversification ultimately trace to evolutionary modifications at the cellular level. This central role of cells frames the basic questions as to how cells work and how cells come to be the way they are. Although these two lines of inquiry lie respectively within the traditional provenance of cell biology and evolutionary biology, a comprehensive synthesis of evolutionary and cell-biological thinking is lacking. We define evolutionary cell biology as the fusion of these two eponymous fields with the theoretical and quantitative branches of biochemistry, biophysics, and population genetics. The key goals are to develop a mechanistic understanding of general evolutionary processes, while specifically infusing cell biology with an evolutionary perspective. The full development of this interdisciplinary field has the potential to solve numerous problems in diverse areas of biology, including the degree to which selection, effectively neutral processes, historical contingencies, and/or constraints at the chemical and biophysical levels dictate patterns of variation for intracellular features. These problems can now be examined at both the within- and among-species levels, with single-cell methodologies even allowing quantification of variation within genotypes. Some results from this emerging field have already had a substantial impact on cell biology, and future findings will significantly influence applications in agriculture, medicine, environmental science, and synthetic biology.

Promoting microtubule assembly: A hypothesis for the functional significance of the +TIP network.

Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machinery that controls MT dynamics remains poorly understood. MT plus-end tracking proteins (+TIPs) are a set of MT-associated proteins that dynamically track growing MT ends and are uniquely positioned to govern MT dynamics. +TIPs associate with each other in a complex array of inter- and intra-molecular interactions known as the "+TIP network." Why do so many +TIPs bind to other +TIPs? Typical answers include the ideas that these interactions localize proteins where they are needed, deliver proteins to the cortex, and/or create regulatory pathways. We propose an additional and more mechanistic hypothesis: that +TIPs bind each other to create a superstructure that promotes MT assembly by constraining the structural fluctuations of the MT tip, thus acting as a polymerization chaperone.

Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin.

Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin's sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmin's minus-end preference results from interactions between stathmin's N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmin's observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

Microtubule assembly dynamics: new insights at the nanoscale.

Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.

Incorporating yeast biosensors into paper-based analytical tools for pharmaceutical analysis.

Paper-based devices serve to address many analytical questions both inside and outside of the laboratory setting. For the first time, yeast is used to construct a whole-cell, paper-based biosensor device. This biologically based paper analytical device (BioPAD) is sensitive to antibiotics in the tetracycline family, and it could potentially address questions of pharmaceutical quality as well as antibiotic contamination in liquids. Our BioPAD can qualitatively discriminate the presence/absence of doxycycline over a range of 30-10,000 µg/mL. In an analysis of a doxycycline dosage form (tablet) commonly used for malaria prophylaxis, BioPADs identified the presence of antibiotic with 92 and 95 % sensitivity, evaluated by eye and computer-assisted image analysis, respectively, with no false positives by either method. BioPADs were found to remain viable for at least 415 days when stored at 4 °C. This research demonstrates the utility of whole yeast cells in paper-based pharmaceutical testing, and it highlights the potential for the development of yeast-based BioPADs to address a range of qualitative analytical questions, especially in low resource settings.

Microtubule dynamic instability: the role of cracks between protofilaments.

Microtubules (MTs) are cytoplasmic protein polymers that are essential for fundamental cellular processes including the maintenance of cell shape, organelle transport and formation of the mitotic spindle. Microtubule dynamic instability is critical for these processes, but it remains poorly understood, in part because the relationship between the structure of the MT tip and the growth/depolymerization transitions is enigmatic. In previous work, we used computational models of dynamic instability to provide evidence that cracks (laterally unbonded regions) between protofilaments play a key role in the regulation of dynamic instability. Here we use computational models to investigate the connection between cracks and dynamic instability in more detail. Our work indicates that while cracks contribute to dynamic instability in a fundamental way, it is not the depth of the cracks per se that governs MT dynamic instability. Instead, what matters more is whether the cracks terminate in GTP-rich or GDP-rich regions of the MT. Based on these observations, we suggest that a functional "GTP cap" (i.e., one capable of promoting MT growth) is one where the cracks terminate in pairs of GTP-bound subunits, and that the likelihood of catastrophe rises significantly with the fraction of crack-terminating subunits that contain GDP. In addition to helping clarify the mechanism of dynamic instability, this idea could also explain how MT stabilizers work: proteins that introduce lateral cross-links between protofilaments would produce islands of GDP-bound tubulin that mimic GTP-rich regions in having strong lateral bonds, thus reducing crack propagation, suppressing catastrophe and promoting rescue.

MTBindingSim: simulate protein binding to microtubules.

SUMMARY: Many protein-protein interactions are more complex than can be accounted for by 1:1 binding models. However, biochemists have few tools available to help them recognize and predict the behaviors of these more complicated systems, making it difficult to design experiments that distinguish between possible binding models. MTBindingSim provides researchers with an environment in which they can rapidly compare different models of binding for a given scenario. It is written specifically with microtubule polymers in mind, but many of its models apply equally well to any polymer or any protein-protein interaction. MTBindingSim can thus both help in training intuition about binding models and with experimental design. AVAILABILITY AND IMPLEMENTATION: MTBindingSim is implemented in MATLAB and runs either within MATLAB (on Windows, Mac or Linux) or as a binary without MATLAB (on Windows or Mac). The source code (licensed under the GNU General Public License) and binaries are freely available at http://mtbindingsim.googlecode.com. CONTACT: jphilip@nd.edu; cpence@nd.edu.

Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe.

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term "stutters." During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.

CLASPing the cell cortex.

CLASPs are spatially regulated microtubule plus end tracking proteins involved in forming polarized microtubule arrays. Work in this issue of Developmental Cell identifies the protein LL5beta as a key CLASP binding platform that mediates communication between the cell cortex and the microtubule cytoskeleton.

Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate.

Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter- molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose: creating a dynamic scaffold that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Here we examine the possibility that this proposed scaffold is a biomolecular condensate (i.e., liquid droplet). Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. Moreover, previous studies have shown that overexpression of the +TIP CLIP-170 induces large "patch" structures containing CLIP-170 and other +TIPs; we hypothesized that these structures might be biomolecular condensates. To test this hypothesis, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.

Characterizing bioavailable phosphorus concentrations in an agricultural stream during hydrologic and streambed disturbances.

In freshwater ecosystems, phosphorus (P) is often considered a growth-limiting nutrient. The use of fertilizers on agricultural fields has led to runoff-driven increases in P availability in streams, and the subsequent eutrophication of downstream ecosystems. Isolated storms and periodic streambed dredging are examples of two common disturbances that contribute dissolved and particulate P to agricultural streams, which can be quantified as soluble reactive P (SRP) using the molybdate-blue method on filtered water samples, or total P (TP) measured using digestions on unfiltered water reflecting all forms of P. While SRP is often considered an approximation of bioavailable P (BAP), research has shown that this is not always the case. Current methods used to estimate BAP do not account for the role of biology (e.g., NaOH extractions) or require specialized platforms (e.g., algal bioassays). Here, in addition to routine analysis of SRP and TP, we used a novel yeast-based bioassay with unfiltered sample water to estimate BAP concentrations during two storms (top 80% and > 95% flow quantiles), and downstream of a reach where management-associated dredging disturbed the streambed. We found that the BAP concentrations were often greater than SRP, suggesting that SRP is not fully representative of P bioavailability. The SRP concentrations were similarly elevated during the two storms, but remained consistently low during streambed disturbance. In contrast, turbidity and TP were elevated during all events. The BAP concentrations were significantly related to turbidity during all disturbance events, but with TP only during storms. The novel yeast assay suggests that BAP export can exceed SRP, particularly when streams are not in equilibrium, such as the rising limb of storms or during active dredging.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

CLIP-170S is a microtubule +TIP variant that confers resistance to taxanes by impairing drug-target engagement.

Taxanes are widely used cancer chemotherapeutics. However, intrinsic resistance limits their efficacy without any actionable resistance mechanism. We have discovered a microtubule (MT) plus-end-binding CLIP-170 protein variant, hereafter CLIP-170S, which we found enriched in taxane-resistant cell lines and patient samples. CLIP-170S lacks the first Cap-Gly motif, forms longer comets, and impairs taxane access to its MT luminal binding site. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Using a computational approach in conjunction with the connectivity map, we unexpectedly discovered that Imatinib was predicted to reverse CLIP-170S-mediated taxane resistance. Indeed, Imatinib treatment selectively depleted CLIP-170S, thus completely reversing taxane resistance. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. Herein, we identify CLIP-170S as a clinically prevalent variant that confers taxane resistance, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials.

Interactions between EB1 and microtubules: dramatic effect of affinity tags and evidence for cooperative behavior.

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.

Syntheses and biological evaluation of ring-C modified colchicine analogs.

Ring-C modified alkaloids were synthesized from colchicine using iminonitroso Diels-Alder reactions in a highly regio- and stereoselective fashion. Several analogs exhibited cytotoxic activity similar to that of colchicine itself against PC-3 and MCF-7 cancer cell lines, by serving as prodrugs of colchicine through retro Diels-Alder reactions under the assayed conditions. In vitro microtubule polymerization assays indicated that these modifications affected their interaction with tubulin.

"Scentsor": A Whole-Cell Yeast Biosensor with an Olfactory Reporter for Low-Cost and Equipment-Free Detection of Pharmaceuticals.

Portable and inexpensive analytical tools are required to monitor pharmaceutical quality in technology limited settings including low- and middle-income countries (LMICs). Whole cell yeast biosensors have the potential to help meet this need. However, most of the readouts for yeast biosensors require expensive equipment or reagents. To overcome this challenge, we have designed a yeast biosensor that produces a unique scent as a readout. This inducible scent biosensor, or "scentsor", does not require the user to administer additional reagents for reporter development and utilizes only the user's nose to be "read". In this Letter, we describe a scentsor that is responsive to the hormone estradiol (E2). The best estimate threshold (BET) for E2 detection with a panel of human volunteers (n = 49) is 39 nM E2 (15 nM when "non-smellers" are excluded). This concentration of E2 is sensitive enough to detect levels of E2 that would be found in dosage forms. This paper provides evidence that scent has the potential for use in portable yeast biosensors as a readout, particularly for use in LMICs.