Precise Spatiotemporal Dynamics Research Articles

Photoactivatable Nanobody Conjugate Dimerizer Temporally Resolves Tiam1-Rac1 Signaling Axis.

The precise spatiotemporal dynamics of protein activities play a crucial role in cell signaling pathways. To control cellular functions in a spatiotemporal manner, a powerful method called photoactivatable chemically induced dimerization (pCID) is used. In this study, photoactivatable nanobody conjugate inducers of dimerization (PANCIDs) is introduced, which combine pCID with nanobody technology. A PANCID consists of a nanobody module that directly binds to an antigenic target, a photocaged small molecule ligand, and a cyclic decaarginine (cR10 *) cell-penetrating peptide (CPP) for efficient nonendocytic intracellular delivery. Therefore, PANCID photodimerizers also benefit from nanobodies, such as their high affinities (in the nm or pm range), specificities, and ability to modulate endogenous proteins. Additionally it is demonstrated that the nanobody moiety can be easily replaced with alternative ones, expanding the potential applications. By using PANCIDs, the dynamics of the Tiam1-Rac1 signaling cascade is investigated and made an interesting finding. It is found that Rac1 and Tiam1 exhibit distinct behaviors in this axis, acting as time-resolved "molecular oscillators" that transit between different functions in the signaling cascade when activated either slowly or rapidly.

Large-scale momentary brain co-activation patterns are associated with hyperalgesia and mediate focal neurochemistry and cross-network functional connectivity in fibromyalgia.

Fibromyalgia has been characterized by augmented cross-network functional communication between the brain's sensorimotor, default mode, and attentional (salience/ventral and dorsal) networks. However, the underlying mechanisms of these aberrant communication patterns are unknown. In this study, we sought to understand large-scale topographic patterns at instantaneous timepoints, known as co-activation patterns (CAPs). We found that a sustained pressure pain challenge temporally modulated the occurrence of CAPs. Using proton magnetic resonance spectroscopy, we found that greater basal excitatory over inhibitory neurotransmitter levels within the anterior insula orchestrated higher cross-network connectivity between the anterior insula and the default mode network through lower occurrence of a CAP encompassing the attentional networks during sustained pain. Moreover, we found that hyperalgesia in fibromyalgia was mediated through increased occurrence of a CAP encompassing the sensorimotor network during sustained pain. In conclusion, this study elucidates the role of momentary large-scale topographic brain patterns in shaping noxious information in patients with fibromyalgia, while laying the groundwork for using precise spatiotemporal dynamics of the brain for the potential development of therapeutics.

Tissue-specific accumulation of pH-sensing phosphatidic acid determines plant stress tolerance.

The signalling lipid phosphatidic acid (PA) is involved in regulating various fundamental biological processes in plants. However, the mechanisms of PA action remain poorly understood because currently available methods for monitoring PA fail to determine the precise spatio-temporal dynamics of this messenger in living cells and tissues of plants. Here, we have developed PAleon, a PA-specific optogenetic biosensor that reports the concentration and dynamics of bioactive PA at the plasma membrane based on Förster resonance energy transfer (FRET). PAleon was sensitive enough to monitor physiological concentrations of PA in living cells and to visualize PA dynamics at subcellular resolution in tissues when they were challenged with abscisic acid (ABA) and salt stress. PAleon bioimaging revealed kinetics and tissue specificity of salt stress-triggered PA accumulation. Compared with wild-type Arabidopsis, the pldα1 mutant lacking phospholipase Dα1 (PLDα1) for PA generation showed delayed and reduced PA accumulation. Comparative analysis of wild type and pldα1 mutant indicated that cellular pH-modulated PA interaction with target proteins and PLD/PA-mediated salt tolerance. Application of the PA biosensor PAleon uncovered specific spatio-temporal PA dynamics in plant tissues. Our findings suggest that PA signalling integrates with cellular pH dynamics to mediate plant response to salt stress.

Imaging the Dynamics of Mitophagy in Live Cells.

Investigating the precise spatiotemporal dynamics of mitophagy can provide insights into how mitochondrial quality control is regulated in different tissues and organisms. Here, we outline live imaging assays to quantitatively assess mitophagy dynamics in real time. This protocol describes both chemical and optogenetic techniques to induce mitochondrial damage with high spatial and temporal control. Using these assays, mitochondria can be tracked from before they sustain damage up to their engulfment by autophagosomes and acidification by lysosomes.

Novel Biosensor Design Reveals the Role and Regulation of GEF-H1 in Cell Migration

Rho family GTPases are molecular switches that control cellular movement. Their activity is regulated with precise spatio-temporal dynamics by guanine exchange factors (GEFs). While biosensors have revealed the dynamics of Rho GTPases in polarized motility, little is known about the subcellular distribution and timing of GEF activation. Guanine exchange factor H1 (GEF-H1) activates RhoA at the leading edge of moving cells and plays a critical role in focal adhesion turnover and mechanosensing. GEF-H1 is unique among GEFs because it is sequestered on microtubules (MTs) in its inactive state and its activation requires microtubule dissociation. GEF-H1 activity is thought to be regulated by MT remodeling and to be essential for crosstalk between the MT and actin networks. We identified a new mode of GEF-H1 regulation and used it to design a fluorescent biosensor that reports GEF-H1 activation in living cells. The biosensor was designed using general structure-based principles that can be extended to the other 68 members of the Dbl family of GEFs. Live cell imaging experiments of GEF-H1 biosensor and MT +TIP labelling in the same cell showed that GEF-H1 is localized at the MTs in its inactive state, and that GEF-H1 was active away from the MTs. We developed a new image analysis method to detect MTs disassembly through detection and tracking of MT +TIPs. We then correlated MT disassembly frequency with GEF-H1 activity in space and time using local windows adaptive to cell membrane fluctuation. Using this techniques, we measured that MT instability regulates GEF-H1 activity at the cell edge with a time delay of 10 seconds. Current studies that investigate the role of kinases in mediating this GEF-H1 activation will be described.

Phosphoinositides: the lipids coordinating cell dynamics

Within the glycerophospholipid family, phosphoinositides, which are minor components of eukaryotic cell membranes, play a critical role as spatiotemporal organizers of cell dynamics. By specifically interacting with proteins, they coordinate the formation and the organization of multiprotein complexes involved in cell signaling, intracellular trafficking and cytoskeleton rearrangement. The highly precise spatiotemporal dynamics of phosphoinositides-regulated mechanisms is ensured by kinases and phosphatases that specifically produce, hydrolyze and control the interconversion of these lipids. The direct implication of these enzymes in human pathologies such as genetic diseases, cancer or infectious pathologies, and the recent arrival of inhibitors targeting some phosphoinositide kinases in clinic, illustrate the mandatory functions of these fascinating lipids.

Signaling from Within: Endocytic Trafficking of the Robo Receptor Is Required for Midline Axon Repulsion.

Signaling from Within: Endocytic Trafficking of the Robo Receptor Is Required for Midline Axon Repulsion.

FRET-Based Genetically Encoded Sensor of Calcineurin Activation

Calcineurin plays a central role in relaying Ca2+ signals from the plasma membrane to the cell nucleus. The resulting involvement of this phosphotase in diseases as diverse as schizophrenia and cardiac hypertrophy necessitates understanding of calcineurin's precise spatio-temporal dynamics in live cells. To this end, genetically encoded FRET-based sensors have shown some potential for elucidating the activity of signaling kinases and phosphatases. However, the currently available calcineurin sensors suffer from limited dynamic range and slow kinetics. Here, we report on the development of a FRET-based sensor of calcinuerin activation (DuoCALN), based on fusions of cerulean and venus to various calcineurin subunits. Such sensors feature improved dynamic range (∼40% change in FRET efficiency upon full activation) and rapid on/off kinetics (∼tens of seconds). These properties are evident in HEK293 cells expressing both DuoCALN and M1 receptors, as summarized in the figure. Bath application of Acetyl-β-methylcholine (horizontal bar) activates these receptors, in turn triggering phasic Ca2+ release from intracellular stores. In response, single-cell FRET efficiency of DuoCALN displays a rapid onset, followed by readily reversible dissipation. The ongoing development of DuoCALN sensors promises new insight into the spatio-temporal dynamics of calcineurin activation in live cells.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Spatio-temporal modelling of the Hes1 and p53-Mdm2 intracellular signalling pathways

The correct localisation of transcription factors is vitally important for the proper functioning of many intracellular signalling pathways. Experimental data has shown that many pathways exhibit oscillations in concentrations of the substances involved, both temporally and spatially. Negative feedback loops are important components of these oscillations, providing fine regulation for the factors involved. In this paper we consider mathematical models of two such pathways—Hes1 and p53-Mdm2. Building on previous mathematical modelling approaches, we derive systems of partial differential equations to capture the evolution in space and time of the variables in the Hes1 and p53-Mdm2 systems. Through computational simulations we show that our reaction-diffusion models are able to produce sustained oscillations both spatially and temporally, accurately reflecting experimental evidence and advancing previous models. The simulations of our models also allow us to calculate a diffusion coefficient range for the variables in each mRNA and protein system, as well as ranges for other key parameters of the models, where sustained oscillations are observed. Finally, by exploiting the explicitly spatial nature of the partial differential equations, we are also able to manipulate mathematically the spatial location of the ribosomes, thus controlling where the proteins are synthesized within the cytoplasm. The results of these simulations predict an optimal distance outside the nucleus where protein synthesis should take place in order to generate sustained oscillations. Using partial differential equation models, new information can be gained about the precise spatio-temporal dynamics of mRNA and proteins. The ability to determine spatial localisation of proteins within the cell is likely to yield fresh insight into a range of cellular diseases such as diabetes and cancer.

A genetically encoded photoactivatable Rac controls the motility of living cells

The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties1 or using photoreactive small molecule ligands2. However, this requires use of toxic UV wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (i.e. through microinjection). We have developed a new approach to produce genetically-encoded photo-activatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics3,4. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin5,6, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458 or 473 nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, while PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicron precision7,8. Their mutual regulation remains controversial9, with data indicating that Rac inhibits and/or activates Rho10,11. Rac was shown to inhibit RhoA in living cells, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response. Rab5 is known as a key regulator of the early endocytic pathway and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.

Spatiotemporal Analysis of Ca2+ Waves in Relation to the Sperm Entry Site and Animal–Vegetal Axis during Ca2+ Oscillations in Fertilized Mouse Eggs

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca2+ concentration ([Ca2+]i) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca2+]i rise were investigated by high-speed Ca2+ imaging during early development of monospermic eggs. Every [Ca2+]i rise involved a Ca2+ wave. In the first Ca2+ transient, [Ca2+]i increased in two steps separated by a “shoulder” point, suggesting two distinct Ca2+ release mechanisms. The first step was a Ca2+ wave that propagated from the sperm-fusion site to its antipode in 4–5 s (velocity, ∼20 μm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca2+]i rise of 12–15 s. A slight cytoplasmic movement followed the Ca2+ wave in the same direction and recovered in 25–35 s. These characteristics changed as follows, as Ca2+ oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation (∼3 h after fertilization). (1) The duration of Ca2+ transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca2+]i rise became greater and wave speeds increased up to 80–100 μm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca2+ wave, although its displacement became smaller. (5) The Ca2+ wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.