Agarose Microchambers for Long-term Calcium Imaging of <em>Caenorhabditis elegans</em>

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Max Planck Institute for Biophysical Chemistry

The motility of molecular motors and the dynamic instability of microtubules are key dynamic processes for mitotic spindle assembly and function. We report here that one of the mitotic kinesins that localizes to chromosomes, Xklp1 from Xenopus laevis, could inhibit microtubule growth and shrinkage. This effect appeared to be mediated by a structural change in the microtubule lattice. We also found that Xklp1 could act as a fast, nonprocessive, plus end-directed molecular motor. The integration of the two properties, motility and inhibition of microtubule dynamics, in one molecule emphasizes the versatile properties of kinesin family members.

A kinesin-like motor inhibits microtubule dynamic instability.

The position of the cytokinesis furrow in a cell determines the relative sizes of its two daughter cells as well as the distribution of their contents. In animal cells, the position of the cytokinesis furrow is specified by the position of the mitotic spindle. The cytokinesis furrow bisects the spindle midway between the microtubule asters, at the site of the microtubule-based midzone, producing two daughter cells. Experiments in some cell types have suggested that the midzone positions the furrow, but experiments in other cells have suggested that the asters position the furrow. One possibility is that different organisms and cell types use different mechanisms to position the cytokinesis furrow. An alternative possibility is that both asters and the midzone contribute to furrow positioning. Recent work in C. elegans has suggested that centrosome separation and the midzone are implicated in cytokinesis. Here we examine the relative contributions of different parts of the mitotic spindle to positioning of the cytokinesis furrow in the C. elegans zygote. By spatially separating the spindle midzone from one of the asters using an ultraviolet laser, we show that the cytokinesis furrow is first positioned by a signal determined by microtubule asters, and then by a second signal that is derived from the spindle midzone. Thus, the position of the cytokinesis furrow is specified by two consecutive furrowing activities.

A cytokinesis furrow is positioned by two consecutive signals.

At the end of the cell cycle a cell physically divides into two daughter cells in a process called cytokinesis. Cytokinesis consists of at least four steps: (1) The position of the presumptive cytokinesis furrow is specified. (2) A contractile ring is formed. (3) The contractile ring contracts, resulting in furrow ingression. (4) Cytokinesis completes with sealing of the membranes. The mitotic spindle positions the cytokinesis furrow at the cell cortex midway along the longitudinal axis of the spindle, which is both the mid-point between the two asters and the location of the spindle midzone. The mitotic spindle emits two consecutive signals that position the furrow: Microtubule asters provide a first signal; the spindle midzone provides a second signal. Our results support the view that the spindle midzone is dispensable for completion of cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis, possibly because the aster- and midzone-positioned furrows compete for contractile elements.

Cytokinesis and the spindle midzone.

At anaphase, the mitotic spindle positions the cytokinesis furrow [1]. Two populations of spindle microtubules are implicated in cytokinesis: radial microtubule arrays called asters and bundled nonkinetochore microtubules called the spindle midzone [2-4]. In C. elegans embryos, these two populations of microtubules provide two consecutive signals that position the cytokinesis furrow: The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone [5]. Evidence for two cytokinesis signals came from the identification of molecules that block midzone-positioned cytokinesis [5-7]. However, no molecules that are only required for, and thus define, the molecular pathway of aster-positioned cytokinesis have been identified. With RNAi screening, we identify LET-99 and the heterotrimeric G proteins GOA-1/GPA-16 and their regulator GPR-1/2 [10-12] in aster-positioned cytokinesis. By using mechanical spindle displacement, we show that the anaphase spindle positions cortical LET-99, at the site of the presumptive cytokinesis furrow. LET-99 enrichment at the furrow depends on the G proteins. GPR-1 is locally reduced at the site of cytokinesis-furrow formation by LET-99, which prevents accumulation of GPR-1 at this site. We conclude that LET-99 and the G proteins define a molecular pathway required for aster-positioned cytokinesis.

LET-99, GOA-1/GPA-16, and GPR-1/2 are required for aster-positioned cytokinesis.

We present a physical analysis of the dynamics and mechanics of contractile actin rings. In particular, we analyze the dynamics of ring contraction during cytokinesis in the Caenorhabditis elegans embryo. We present a general analysis of force balances and material exchange and estimate the relevant parameter values. We show that on a microscopic level contractile stresses can result from both the action of motor proteins, which cross-link filaments, and from the polymerization and depolymerization of filaments in the presence of end-tracking cross-linkers.

Stress generation and filament turnover during actin ring constriction.

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

Mechanical and genetic separation of aster- and midzone-positioned cytokinesis.

In asymmetrically dividing cells, a failure to coordinate cell polarity with the site of cell division can lead to cell fate transformations and tumorigenesis. Cell polarity in C. elegans embryos is defined by PAR proteins, which occupy reciprocal halves of the cell cortex. During asymmetric division, the boundary between the anterior and posterior PAR domains precisely matches the site of cell division, ensuring exclusive segregation of cell fate. The PAR domains determine the site of cell division by positioning the mitotic spindle, suggesting one means by which cell polarity and cell division might be coordinated. Here, we report that cell polarity and cell division are coordinated through an additional mechanism: the site of cell division repositions the PAR-2 boundary. Galpha-mediated microtubule-cortex interactions appear to direct cortical flows of PAR-2 and myosin toward the site of cell division, which acts as a PAR-2 and myosin sink. Embryos with defects in PAR-2 boundary correction undergo mis-segregation of cortical polarity and cytoplasmic determinants, suggesting that PAR domain correction might help prevent cell fate transformation.

Cortical domain correction repositions the polarity boundary to match the cytokinesis furrow in C. elegans embryos.

We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter.

Codon adaptation-based control of protein expression in C. elegans.

Caenorhabditis elegans larvae display specific behavior and development that is not observed in adults. For example, larvae go through a molting cycle that includes a sleep-like state prior to the molt. The study of these processes requires high-resolution long-term observation of individual animals. Here we describe a method for simultaneous culture and observation of several individual young C. elegans larvae inside agarose hydrogel-based arrayed microcompartments. We used agarose hydrogel microcompartments to observe and quantify larval specific sleep-wake-like behavior and to observe neuronal rewiring using confocal fluorescence microscopy without acute immobilization. We found no behavioral aberrations caused by area restriction. We show that worms cultured inside hydrogel microcompartments develop into normal adults. Thus, hydrogel microcompartments appear useful for long-term observation of larval behavior and development.

Agarose hydrogel microcompartments for imaging sleep- and wake-like behavior and nervous system development in Caenorhabditis elegans larvae.

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Reducci&oacute;n de la actividad de una neurona sensorial durante un estado similar al sue&ntilde;o en Caenorhabditis elegans.

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Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans.

R&eacute;duction de l&#39;activit&eacute; d&#39;un neurone sensitif durant un &eacute;tat semblable au sommeil chez Caenorhabditis elegans.

Verminderte Aktivit&auml;t eines sensorischen Neurons w&auml;hrend einem Schlaf-&auml;hnlichen Zustand in Caenorhabditis Elegans.

Ridotta attivit&agrave; di un neurone sensoriale durante uno stato di sonno in Caenorhabditis elegans.

Cytokinesis and spindle positioning require the cortical force regulator G Protein Regulator 1/2 (GPR-1/2). GPR-1/2 is thought to localize to sites of cortical force generation. Does GPR-1/2 also act as a sensor for mechanical stimulation? I mechanically stimulated the cortex by indenting it with a glass needle and observed the cortical localization of a YFP::GPR-1 transgene. I found that cortical YFP::GPR-1 accumulated at the site of mechanical indentation. This phenomenon occurred on most of the cortical areas except the site of prospective cytokinesis furrow formation. This result suggests that GPR-1/2 can sense mechanical properties of the cortex, which may be important for GPR-1/2 function regulating spindle positioning and cytokinesis.

G protein regulator 1 (GPR-1) localizes to cortical sites of artificial mechanical indentation in Caenorhabditis elegans zygotes.

Sleep is characterized by reduced muscle activity resulting in reduced movement and a typical posture compatible with relaxed muscles. Prior to each molt, C. elegans larvae go through a phase of behavioral quiescence called Lethargus. Lethargus has sleep-like properties, but a specific posture has not yet been described. Do C. elegans larvae relax their muscles during sleep and do they assume a typical posture? We measured worm posture and body wall muscle activity using calcium imaging across the sleep-wake-like cycle. We found that worms were less curved and had less muscle activity during the sleep-like state. We conclude that during Lethargus, muscle activity is reduced, resulting in a relaxed body posture typical for a sleep-like state.

Reduced muscle contraction and a relaxed posture during sleep-like Lethargus.

Sleep-like states are characterized by massively reduced behavioral activity. Little is known about genetic control of sleep-like behavior. It is also not clear how general activity levels during wake-like behavior influence activity levels during sleep-like behavior. Mutations that increase wake-like activity are generally believed to also increase activity during sleep-like behavior and mutations that decrease wake-like activity are believed to have decreased activity during sleep-like behavior. We studied sleep-like behavior during lethargus in larvae of Caenorhabditis elegans. We looked through a small set of known mutants with altered activity levels. As expected, mutants with increased activity levels typically showed less sleep-like behavior. Among these hyperactive mutants was a gain-of-function mutant of the conserved heterotrimeric G protein subunit Galphaq gene egl-30. We found, however, that an unusual semidominant hypoactive mutant of egl-30 also had reduced sleep-like behavior. While movement was severely reduced and impaired in the semidominant egl-30 mutant, sleep-like behavior was severely reduced: the semidominant egl-30 mutant lacked prolonged periods of complete immobility, reduced spontaneous neural activity less, and reduced responsiveness to stimulation less. egl-30 is a well-known regulator of behavior. Our results suggest that egl-30 controls not only general activity levels, but also differences between wake-like and sleep-like behavior.

Reduced sleep-like quiescence in both hyperactive and hypoactive mutants of the Galphaq Gene egl-30 during lethargus in Caenorhabditis elegans.

Sleep is an essential behavior that is found in all animals that have a nervous system. Neural activity is thought to control sleep, but little is known about the identity and the function of neural circuits underlying sleep. Lethargus is a developmentally regulated period of behavioral quiescence in C. elegans larvae that has sleep-like properties.

An AP2 transcription factor is required for a sleep-active neuron to induce sleep-like quiescence in C. elegans.

During their development, Caenorhabditis elegans larvae go through four developmental stages. At the end of each larval stage, nematodes molt. They synthesize a new cuticle and shed the old cuticle. During the molt, larvae display a sleep-like behavior that is called lethargus. We wanted to determine how gene expression changes during the C. elegans molting cycle. We performed transcriptional profiling of C. elegans by selecting larvae displaying either sleep-like behavior during the molt or wake behavior during the intermolt to identify genes that oscillate with the molting-cycle. We found that expression changed during the molt and we identified 520 genes that oscillated with the molting cycle. 138 of these genes were not previously reported to oscillate. The majority of genes that had oscillating expression levels appear to be involved in molting, indicating that the majority of transcriptional changes serve to resynthesize the cuticle. Identification of genes that control sleep-like behavior during lethargus is difficult but may be possible by looking at genes that are expressed in neurons. 22 of the oscillating genes were expressed in neurons. One of these genes, the dopamine transporter gene dat-1, was previously shown in mammals and in C. elegans to control sleep. Taken together, we provide a dataset of genes that oscillate with the molting and sleep-wake cycle, which will be useful to investigate molting and possibly also sleep-like behavior during lethargus.

Henrik Bringmann

Max Planck Institute for Biophysical Chemistry

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans