PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. A microtubule-localized proximity biotinylation tool revealed that depletion of PLEKHA5 decreased the extent of APC/C association with microtubules. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

Sphingosine-1-Phosphate Signaling in Ischemic Stroke: From Bench to Bedside and Beyond

Sphingosine-1-phosphate (S1P) signaling is being increasingly recognized as a strong modulator of immune cell migration and endothelial function. Fingolimod and other S1P modulators in ischemic stroke treatment have shown promise in emerging experimental models and small-scale clinical trials. In this article, we will review the current knowledge of the role of S1P signaling in brain ischemia from the aspects of inflammation and immune interventions, sustaining endothelial functions, regulation of blood-brain barrier integrity, and functional recovery. We will then discuss the current and future therapeutic perspectives of targeting S1P for the treatment of ischemic stroke. Mechanism studies would help to bridge the gap between preclinical studies and clinical practice. Future success of bench-to-bedside translation shall be based on in depth understanding of S1P signaling during stroke and on the ability to have a fine temporal and spatial regulation of the signal pathway.

Microtubule Lattice Spacing Governs Cohesive Envelope Formation of Tau Family Proteins

Tau is an intrinsically-disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive envelopes around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here, we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau-envelope disassembly. Investigating other members of the tau-MAP family, we find MAP2 similarly forms envelopes governed by lattice-spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct segments. We conclude that the interdependent allostery between lattice-spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

Patterning with clocks and genetic cascades: Segmentation and regionalization of vertebrate versus insect body plans

Oscillatory and sequential processes have been implicated in the spatial patterning of many embryonic tissues. For example, molecular clocks delimit segmental boundaries in vertebrates and insects and mediate lateral root formation in plants, whereas sequential gene activities are involved in the specification of regional identities of insect neuroblasts, vertebrate neural tube, vertebrate limb, and insect and vertebrate body axes. These processes take place in various tissues and organisms, and, hence, raise the question of what common themes and strategies they share. In this article, we review 2 processes that rely on the spatial regulation of periodic and sequential gene activities: segmentation and regionalization of the anterior–posterior (AP) axis of animal body plans. We study these processes in species that belong to 2 different phyla: vertebrates and insects. By contrasting 2 different processes (segmentation and regionalization) in species that belong to 2 distantly related phyla (arthropods and vertebrates), we elucidate the deep logic of patterning by oscillatory and sequential gene activities. Furthermore, in some of these organisms (e.g., the fruit fly Drosophila), a mode of AP patterning has evolved that seems not to overtly rely on oscillations or sequential gene activities, providing an opportunity to study the evolution of pattern formation mechanisms.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Abstract P461: Phospho-regulated Spatial Regulation Of α-tat1 Mediates Dynamic Microtubule Acetylation

Microtubules (MTs) provide mechanical strength to cardiomyocytes and dysregulation of MT network is implicated in cardiac diseases. MT acetylation mediates mechanotransduction, provides structural flexibility to cardiomyocytes and protects against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, whose only known substrate is α-tubulin in polymerized MTs. However, little is known about how α-TAT1 itself is regulated. Here we report that intracellular spatial localization of α-TAT1 mediates MT acetylation. Specifically, we identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import and cytosolic retention. Inhibition of Exportin 1-mediated nuclear export induced nuclear accumulation of α-TAT1 and loss of MT acetylation. We found that α-TAT1 nuclear localization is inhibited by CDKs, CK2 and PKA kinases, pharmacological inhibition of which increased nuclear localization of α-TAT1 and inhibited MT acetylation. We identified a critical phosphoThreonine (T 322 ) that binds to 14-3-3 proteins (β, γ, ε and ζ isoforms) downstream of kinases and mediates cytosolic retention of α-TAT1. Inhibition of 14-3-3 proteins also increased nuclear accumulation of α-TAT1. Fibroblastic cells expressing a phosphodeficient α-TAT1 (T322A) show defects in DNA damage response and increased cell proliferation, which may be pertinent to cardiac hypertrophy. Based on these observations, we developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from the nucleus to the cytosol on blue light stimulation. HeLa cells expressing optoATAT1 exposed to light showed increased MT acetylation unlike those kept in dark, validating the tool. In summary, we have identified a novel role for the C-terminal region of α-TAT1 in regulating its function through dynamic intracellular localization downstream of kinases and 14-3-3 proteins. We have identified multiple pharmacological agents to modulate MT acetylation through spatial regulation of α-TAT1. We have also developed an optogenetic tool to control MT acetylation that will help in elucidating the role of MT acetylation in disease states.