Karl Emanuel Busch

The Schizosaccharomyces pombe EB1 Homolog Mal3p Binds and Stabilizes the Microtubule Lattice Seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

The Microtubule Plus End-Tracking Proteins mal3p and tip1p Cooperate for Cell-End Targeting of Interphase Microtubules

BACKGROUND CLIP-170 and EB1 protein family members localize to growing microtubule tips and link spatial information with the control of microtubule dynamics. It is unknown whether these proteins operate independently or whether their actions are coordinated. In fission yeast the CLIP-170 homolog tip1p is required for targeting of microtubules to cell ends, whereas the role of the EB1 homolog mal3p in microtubule organization has not been investigated. RESULTS We show that mal3p promotes the initiation of microtubule growth and inhibits catastrophes. Premature catastrophes occur randomly throughout the cell in the absence of mal3p. mal3p decorates the entire microtubule lattice and localizes to particles along the microtubules and at their growing tips. Particles move in two directions, outbound toward the cell ends or inbound toward the cell center. At cell ends, the microtubule tip-associated mal3p particles disappear followed by a catastrophe. mal3p localizes normally in tip1-deleted cells and disappears from microtubule tips preceding the premature catastrophes. In contrast, tip1p requires mal3p to localize at microtubule tips. mal3p and tip1p directly interact in vitro. CONCLUSIONS mal3p and tip1p form a system allowing microtubules to target cell ends. We propose that mal3p stimulates growth initiation and maintains growth by suppressing catastrophes. At cell ends, mal3p disappears from microtubule tips followed by a catastrophe. mal3p is involved in recruiting tip1p to microtubule tips. This becomes important when microtubules contact the cell cortex outside the cell ends because mal3p dissociates prematurely without tip1p, which is followed by a premature catastrophe.

A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans

Homeostasis of internal carbon dioxide (CO2) and oxygen (O2) levels is fundamental to all animals. Here we examine the CO2 response of the nematode Caenorhabditis elegans. This species inhabits rotting material, which typically has a broad CO2 concentration range. We show that well fed C. elegans avoid CO2 levels above 0.5%. Animals can respond to both absolute CO2 concentrations and changes in CO2 levels within seconds. Responses to CO2 do not reflect avoidance of acid pH but appear to define a new sensory response. Sensation of CO2 is promoted by the cGMP-gated ion channel subunits TAX-2 and TAX-4, but other pathways are also important. Robust CO2 avoidance in well fed animals requires inhibition of the DAF-16 forkhead transcription factor by the insulin-like receptor DAF-2. Starvation, which activates DAF-16, strongly suppresses CO2 avoidance. Exposure to hypoxia (<1% O2) also suppresses CO2 avoidance via activation of the hypoxia-inducible transcription factor HIF-1. The npr-1 215V allele of the naturally polymorphic neuropeptide receptor npr-1, besides inhibiting avoidance of high ambient O2 in feeding C. elegans, also promotes avoidance of high CO2. C. elegans integrates competing O2 and CO2 sensory inputs so that one response dominates. Food and allelic variation at NPR-1 regulate which response prevails. Our results suggest that multiple sensory inputs are coordinated by C. elegans to generate different coherent foraging strategies.

Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans

Behaviours evolve by iterations of natural selection, but we have few insights into the molecular and neural mechanisms involved. Here we show that some Caenorhabditis elegans wild strains switch between two foraging behaviours in response to subtle changes in ambient oxygen. This finely tuned switch is conferred by a naturally variable hexacoordinated globin, GLB-5. GLB-5 acts with the atypical soluble guanylate cyclases, which are a different type of oxygen binding protein, to tune the dynamic range of oxygen-sensing neurons close to atmospheric (21%) concentrations. Calcium imaging indicates that one group of these neurons is activated when oxygen rises towards 21%, and is inhibited as oxygen drops below 21%. The soluble guanylate cyclase GCY-35 is required for high oxygen to activate the neurons; GLB-5 provides inhibitory input when oxygen decreases below 21%. Together, these oxygen binding proteins tune neuronal and behavioural responses to a narrow oxygen concentration range close to atmospheric levels. The effect of the glb-5 gene on oxygen sensing and foraging is modified by the naturally variable neuropeptide receptor npr-1 (refs 4, 5), providing insights into how polygenic variation reshapes neural circuit function.

Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior

Summary Homeostatic control of body fluid CO2 is essential in animals but is poorly understood. C. elegans relies on diffusion for gas exchange and avoids environments with elevated CO2. We show that C. elegans temperature, O2, and salt-sensing neurons are also CO2 sensors mediating CO2 avoidance. AFD thermosensors respond to increasing CO2 by a fall and then rise in Ca2+ and show a Ca2+ spike when CO2 decreases. BAG O2 sensors and ASE salt sensors are both activated by CO2 and remain tonically active while high CO2 persists. CO2-evoked Ca2+ responses in AFD and BAG neurons require cGMP-gated ion channels. Atypical soluble guanylate cyclases mediating O2 responses also contribute to BAG CO2 responses. AFD and BAG neurons together stimulate turning when CO2 rises and inhibit turning when CO2 falls. Our results show that C. elegans senses CO2 using functionally diverse sensory neurons acting homeostatically to minimize exposure to elevated CO2.

Neuronal and molecular substrates for optimal foraging in Caenorhabditis elegans

Variation in food quality and abundance requires animals to decide whether to stay on a poor food patch or leave in search of better food. An important question in behavioral ecology asks when is it optimal for an animal to leave a food patch it is depleting. Although optimal foraging is central to evolutionary success, the neural and molecular mechanisms underlying it are poorly understood. Here we investigate the neuronal basis for adaptive food-leaving behavior in response to resource depletion in Caenorhabditis elegans, and identify several of the signaling pathways involved. The ASE neurons, previously implicated in salt chemoattraction, promote food-leaving behavior via a cGMP pathway as food becomes limited. High ambient O2 promotes food-leaving via the O2-sensing neurons AQR, PQR, and URX. Ectopic activation of these neurons using channelrhodopsin is sufficient to induce high food-leaving behavior. In contrast, the neuropeptide receptor NPR-1, which regulates social behavior on food, acts in the ASE neurons, the nociceptive ASH neurons, and in the RMG interneuron to repress food-leaving. Finally, we show that neuroendocrine signaling by TGF-β/DAF-7 and neuronal insulin signaling are necessary for adaptive food-leaving behavior. We suggest that animals integrate information about their nutritional state with ambient oxygen and gustatory stimuli to formulate optimal foraging strategies.

Neuronal Control of Metabolism through Nutrient-Dependent Modulation of Tracheal Branching

Summary During adaptive angiogenesis, a key process in the etiology and treatment of cancer and obesity, the vasculature changes to meet the metabolic needs of its target tissues. Although the cues governing vascular remodeling are not fully understood, target-derived signals are generally believed to underlie this process. Here, we identify an alternative mechanism by characterizing the previously unrecognized nutrient-dependent plasticity of the Drosophila tracheal system: a network of oxygen-delivering tubules developmentally akin to mammalian blood vessels. We find that this plasticity, particularly prominent in the intestine, drives—rather than responds to—metabolic change. Mechanistically, it is regulated by distinct populations of nutrient- and oxygen-responsive neurons that, through delivery of both local and systemic insulin- and VIP-like neuropeptides, sculpt the growth of specific tracheal subsets. Thus, we describe a novel mechanism by which nutritional cues modulate neuronal activity to give rise to organ-specific, long-lasting changes in vascular architecture.

Calcium dynamics regulating the timing of decision-making in C. elegans

Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca2+]i), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+]i rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making. DOI: http://dx.doi.org/10.7554/eLife.21629.001

Should I stay or should I go

Most animals inhabit environments in which resources are heterogeneous and distributed in patches. A fundamental question in behavioral ecology is how an animal feeding on a particular food patch, and hence depleting it, decides when it is optimal to leave the patch in search of a richer one. Optimal foraging has been extensively studied and modeled in animals not amenable to molecular and neuronal manipulation. Recently, however, we and others have begun to elucidate at a mechanistic level how food patch leaving decisions are made. We found that C. elegans leaves food with increasing probability as food patches become depleted. Therefore, despite its artificial laboratory environment, its behavior conforms to the optimal foraging theory, which allowed us to genetically dissect the behavior. Here we expand our discussion on some of these findings, in particular how metabolism, oxygen and carbon dioxide regulate C. elegans food leaving behavior.