SnapShot: Mechanical Forces in Development I.

Cell-type-specific F-actin structures and myosin motors are key generators of the forces that drive tissue morphogenesis in developing organisms. These cytoskeletal elements mediate defined cell deformation and control the arrangement of cell-cell contacts. This SnapShot presents a selection of morphogenetic processes, the analysis of which has pioneered specific types of F-actin/myosin-mediated force generation in development.

SnapShot: Mechanical Forces in Development II.

The forces shaping an organism are not exclusively produced by actin/myosin II networks. In part II of this SnapShot, we present various alternative mechanisms. In addition to driving morphogenesis, cells use mechanical forces to sense and react to the specific mechanical properties of their environment. Also, we present a selection of experimental tools commonly used in force analysis.

Sterol-Rich Membrane Domains Define Fission Yeast Cell Polarity.

Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.

Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure.

Dorsal closure is a tissue-modeling process in the developing Drosophila embryo during which an epidermal opening is closed. It begins with the appearance of a supracellular actin cable that surrounds the opening and provides a contractile force. Amnioserosa cells that fill the opening produce an additional critical force pulling on the surrounding epidermal tissue. We show that this force is not gradual but pulsed and occurs long before dorsal closure starts. Quantitative analysis, combined with laser cutting experiments and simulations, reveals that tension-based dynamics and cell coupling control the force pulses. These constitutively pull the surrounding epidermal tissue dorsally, but the displacement is initially transient. It is translated into dorsal-ward movement only with the help of the actin cable, which acts like a ratchet, counteracting ventral-ward epidermis relaxation after force pulses. Our work uncovers a sophisticated mechanism of cooperative force generation between two major forces driving morphogenesis.

Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure.

The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. Our patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. Here, we used this tool to quantify the in vivo force production of Myosin II waves that we observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing for the first time to our knowledge quantitative values on an active Myosin-driven force, we elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and we characterized the mechanical properties of the dorsal yolk cell surface.

Reversible solidification of fission yeast cytoplasm after prolonged nutrient starvation.

Cells depend on a highly ordered organisation of their content and must develop strategies to maintain the anisotropic distribution of organelles during periods of nutrient shortage. One of these strategies is to solidify the cytoplasm, which was observed in bacteria and yeast cells with acutely interrupted energy production. Here, we describe a different type of cytoplasm solidification fission yeast cells switch to, after having run out of nutrients during multiple days in culture. It provides the most profound reversible cytoplasmic solidification of yeast cells described to date. Our data exclude the previously proposed mechanisms for cytoplasm solidification in yeasts and suggest a mechanism that immobilises cellular components in a size-dependent manner. We provide experimental evidence that, in addition to time, cells use intrinsic nutrients and energy sources to reach this state. Such cytoplasmic solidification may provide a robust means to protect cellular architecture in dormant cells.

Structural analysis of multicellular organisms with cryo-electron tomography.

We developed a method for visualizing tissues from multicellular organisms using cryo-electron tomography. Our protocol involves vitrifying samples with high-pressure freezing, thinning them with cryo-FIB-SEM (focused-ion-beam scanning electron microscopy) and applying fiducial gold markers under cryogenic conditions to the lamellae post-milling. We applied this protocol to acquire tomograms of vitrified Caenorhabditis elegans embryos and worms, which showed the intracellular organization of selected tissues at particular developmental stages in otherwise intact specimens.

Gamma-tubulin complex-mediated anchoring of spindle microtubules to spindle-pole bodies requires Msd1 in fission yeast.

The anchoring of microtubules to subcellular structures is critical for cell polarity and motility. Although the process of anchoring cytoplasmic microtubules to the centrosome has been studied in some detail, it is not known how spindle microtubules are anchored to the mitotic centrosome and, particularly, whether anchoring and nucleation of mitotic spindles are functionally separate. Here, we show that a fission yeast coiled-coil protein, Msd1, is required for anchoring the minus end of spindle microtubules to the centrosome equivalent, the spindle-pole body (SPB). msd1 deletion causes spindle microtubules to abnormally extend beyond SPBs, which results in chromosome missegregation. Importantly, this protruding spindle is phenocopied by the amino-terminal deletion mutant of Alp4, a component of the gamma-tubulin complex (gamma-TuC), which lacks the potential Msd1-interacting domain. We propose that Msd1 interacts with gamma-TuC, thereby specifically anchoring the minus end of microtubules to SPBs without affecting microtubule nucleation.

Cell polarity in fission yeast: a matter of confining, positioning, and switching growth zones.

The two key processes in growth polarisation are the generation of a confined region and the correct positioning of that region. Fission yeast has greatly contributed to the study of cell polarisation, particularly in the aspect of growth site positioning, which involves the interphase microtubule cytoskeleton. Here we review the mechanisms of growth polarity in vegetatively growing fission yeast cells. These seemingly simple cells show astonishingly complex growth polarity behaviour, including polarity switching and integrating multiple levels of control by the cell cycle machinery. We aim to extract and highlight the underlying concepts and discuss these in context of current understanding; showing how relevant proteins are networked to integrate the various machineries.

Electron tomography reveals a flared morphology on growing microtubule ends.

Microtubules (MTs) exhibit dynamic instability, alternating between phases of growth and shortening, mostly at their uncapped plus ends. Based on results from cryo-electron microscopy it was proposed that growing MTs display mainly curved sheets and blunt ends; during depolymerisation curled 'ramshorns' predominate. Observations of MTs in mitotic cells have suggested that the situation in vivo differs from that in vitro, but so far, a clear comparison between in vivo and in vitro results has not been possible because MT end structures could not be correlated directly with the dynamic state of that particular MT. Here we combine light microscopy and electron tomography (ET) to show that growing MT plus ends in the fission yeast Schizosaccharomyces pombe display predominantly a flared morphology. This indicates that MT polymerisation in vivo and in vitro can follow different paths.

Reconstitution of a microtubule plus-end tracking system in vitro.

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila melanogaster.

There is emerging evidence that microtubules in nondividing cells can be employed to remodel the intracellular space. Here, we demonstrate an essential role for microtubules in dorsal closure, which occurs toward the end of Drosophila melanogaster embryogenesis. Dorsal closure is a morphogenetic process similar to wound healing, whereby a gap in the epithelium is closed through the coordinated action of different cell types. Surprisingly, this complex process requires microtubule function exclusively in epithelial cells and only for the last step, the zippering, which seals the gap. Preceding zippering, the epithelial microtubules reorganize to attain an unusual spatial distribution, which we describe with subcellular resolution in the intact, living organism. We provide a clearly defined example where cells of a developing organism transiently reorganize their microtubules to fulfill a specialized morphogenetic task.

Force- and length-dependent catastrophe activities explain interphase microtubule organization in fission yeast.

The cytoskeleton is essential for the maintenance of cell morphology in eukaryotes. In fission yeast, for example, polarized growth sites are organized by actin, whereas microtubules (MTs) acting upstream control where growth occurs. Growth is limited to the cell poles when MTs undergo catastrophes there and not elsewhere on the cortex. Here, we report that the modulation of MT dynamics by forces as observed in vitro can quantitatively explain the localization of MT catastrophes in Schizosaccharomyces pombe. However, we found that it is necessary to add length-dependent catastrophe rates to make the model fully consistent with other previously measured traits of MTs. We explain the measured statistical distribution of MT-cortex contact times and re-examine the curling behavior of MTs in unbranched straight tea1Delta cells. Importantly, the model demonstrates that MTs together with associated proteins such as depolymerizing kinesins are, in principle, sufficient to mark the cell poles.

Force- and kinesin-8-dependent effects in the spatial regulation of fission yeast microtubule dynamics.

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length-dependent increase in the catastrophe frequency that is mediated by kinesin-8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin-8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.

How to grab a microtubule on the move.

In migrating cells, Rho family GTPases and their effectors play a central role in polarizing and in organizing the actin and microtubule cytoskeletons. A study by Fukata et al. in the June 28th issue of Cell now shows that the Rac1/Cdc42 effector IQGAP1 captures microtubules by binding to CLIP170.

Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules.

The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends. The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics. Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips. There, tea2p and tip1p accumulate in larger particles. Particle formation requires the EB1 homolog, mal3p. Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.

Glucose starvation triggers filamentous septin assemblies in an S. pombe septin-2 deletion mutant.

Using correlative light and electron microscopy (CLEM), we studied the intracellular organization by of glucose-starved fission yeast cells (Schizosaccharomyces pombe) with regards to the localization of septin proteins throughout the cytoplasm. Thereby, we found that for cells carrying a deletion of the gene encoding septin-2 (spn2Δ), starvation causes a GFP-tagged version of septin-3 (spn3-GFP) and family members, to assemble into a single, prominent filamentous structure. It was previously shown that during exponential growth, spn2Δ cells form septin-3 polymers. However, the polymers we observed during exponential growth are different from the spn3p-GFP structure we observed in starved cells. Using CLEM, in combination with anti-GFP immunolabeling on plastic-sections, we could assign spn3p-GFP to the filaments we have found in EM pictures. Besides septin-3, these filamentous assemblies most likely also contain septin-1 as an RFP-tagged version of this protein forms a very similar structure in starved spn2Δ cells. Our data correlate phase-contrast and fluorescence microscopy with electron micrographs of plastic-embedded cells, and further on with detailed views of tomographic 3D reconstructions. Cryo-electron microscopy of spn2Δ cells in vitrified sections revealed a very distinct overall morphology of the spn3p-GFP assembly. The fine-structured, regular density pattern suggests the presence of assembled septin-3 filaments that are clearly different from F-actin bundles. Furthermore, we found that starvation causes substantial mitochondria fission, together with massive decoration of their outer membrane by ribosomes.

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.

Amnioserosa cell constriction but not epidermal actin cable tension autonomously drives dorsal closure.

Tissue morphogenesis requires coordination of multiple force-producing components. During dorsal closure in fly embryogenesis, an epidermis opening closes. A tensioned epidermal actin/MyosinII cable, which surrounds the opening, produces a force that is thought to combine with another MyosinII force mediating apical constriction of the amnioserosa cells that fill the opening. A model proposing that each force could autonomously drive dorsal closure was recently challenged by a model in which the two forces combine in a ratchet mechanism. Acute force elimination via selective MyosinII depletion in one or the other tissue shows that the amnioserosa tissue autonomously drives dorsal closure while the actin/MyosinII cable cannot. These findings exclude both previous models, although a contribution of the ratchet mechanism at dorsal closure onset remains likely. This shifts the current view of dorsal closure being a combinatorial force-component system to a single tissue-driven closure event.

The fission yeast MO25 protein functions in polar growth and cell separation.

Proteins of the MO25 family are widely conserved but their function has not been characterized in detail. Human MO25 is a cofactor of LKB1, a conserved protein kinase with roles in cell polarity in nematodes, flies and mammalian cells. Furthermore, the budding yeast MO25 homologue, Hym1, is important for cell separation and morphogenesis. We have characterized Pmo25p, the MO25 homologue in the fission yeast Schizosaccharomyces pombe. Pmo25p is an essential protein required for polar growth; in its absence the actin cytoskeleton becomes depolarized and cells adopt a round morphology. In addition, pmo25 mutants are defective in cell separation. Both functions of Pmo25p appear to be mediated by the Orb6p-Mob2p kinase complex. Pmo25p shows no distinct localization during interphase, but it is recruited to one of the two spindle pole bodies during anaphase and to the division site during cytokinesis. The septation initiation network (SIN) regulates the localization of Pmo25p, suggesting that it regulates Pmo25p function during cell division.