Spindle function in<i>Xenopus</i>oocytes involves possible nanodomain calcium signaling

Li, Ruizhen; Leblanc, Julie; He, Kevin; Liu, X. Johné

doi:10.1091/mbc.e16-05-0338

Cited by 16 publications

(26 citation statements)

References 61 publications

(100 reference statements)

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“…While 10 03BCM free calcium does not persist in cells for long periods of time, and does not occur uniformly throughout the cell, the level of calcium may rise to this level transiently and locally in nanodomains. [99]…”

Section: Discussionmentioning

confidence: 99%

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

Hovey

Fowler

Mahling

et al. 2017

Biophysical Chemistry

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Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3 nM) and (Ca2+)4-CaM (~85 nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2|Qp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

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Section: Discussionmentioning

confidence: 99%

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

Hovey

Fowler

Mahling

et al. 2017

Biophysical Chemistry

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“…To determine whether Xenopus egg meiotic spindles are dependent on calcium nanodomains 10 , TubeCamp was co-expressed with mCh-EMTB (Fig. 2A) or rhodamine-tubulin (not shown).…”

Section: Figurementioning

confidence: 99%

“…However, further studies led to the conclusion that elevation of [Ca 2+ ] i either has no role in these events, plays a permissive role in these events, or functions as an auxiliary signaling pathway that supplements other mechanisms 7 . One potential explanation of the controversy is that specific M-phase events might depend on highly localized increases in [Ca 2+ ] i , variously referred to as microdomains 8 or nanodomains 9 , as proposed recently 10 . Such domains are hypothesized to arise from rapid shuttling of calcium between closely positioned sources and sinks, rendering them potentially difficult to detect with traditional dyes and largely insensitive to slow chelators such as EGTA 9 .…”

mentioning

confidence: 99%

“…Such domains are hypothesized to arise from rapid shuttling of calcium between closely positioned sources and sinks, rendering them potentially difficult to detect with traditional dyes and largely insensitive to slow chelators such as EGTA 9 . Here a novel microtubule-binding calcium sensor—TubeCamp--was used to test the hypothesis 10 that spindles are associated with calcium nanodomains. TubeCamp imaging revealed that spindles in Xenopus eggs, Xenopus embryos, and HeLa cells were all associated with calcium nanodomains at the spindle poles.…”

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confidence: 99%

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Calcium nanodomains in spindles

Mo¹,

Swider²,

Tao³

et al. 2018

Preprint

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37The role of calcium signaling in specific events of animal cell meiosis or mitosis (M-38 phase) is a subject of enduring controversy. Early efforts suggested that increases in 39 intracellular free calcium ([Ca 2+ ] i ) promote spindle disassembly 1, 2 while subsequent 40 work suggested that global [Ca 2+ ] i increases trigger nuclear envelope breakdown, spindle 41 assembly, the metaphase-anaphase transition, and cytokinesis 3-6 . However, further 42 studies led to the conclusion that elevation of [Ca 2+ ] i either has no role in these events, 43 plays a permissive role in these events, or functions as an auxiliary signaling pathway 44 that supplements other mechanisms 7 . One potential explanation of the controversy is 45 that specific M-phase events might depend on highly localized increases in [Ca 2+ ] i , 46 variously referred to as microdomains 8 or nanodomains 9 , as proposed recently 10 . Such 47 domains are hypothesized to arise from rapid shuttling of calcium between closely 48 positioned sources and sinks, rendering them potentially difficult to detect with 49 traditional dyes and largely insensitive to slow chelators such as EGTA 9 . Here a novel 50 microtubule-binding calcium sensor-TubeCamp--was used to test the hypothesis 10 that 51 spindles are associated with calcium nanodomains. TubeCamp imaging revealed that 52 spindles in Xenopus eggs, Xenopus embryos, and HeLa cells were all associated with 53 calcium nanodomains at the spindle poles. Calcium nanodomains also formed in spindles 54 assembled in cell extracts and at the center of monopolar spindles, suggesting that they 55 are a basic feature of spindle self-assembly. Disruption of calcium nanodomains via 56 perturbation of inositol-1,4,5-trisphosphate signaling or rapid chelation of [Ca 2+ ]i resulted 57 in spindle disassembly in vivo and vitro. The results demonstrate the existence of 58 spindle-associated calcium nanodomains and indicate that such domains are an essential 59 and common feature of spindles in vertebrates. 60 61 To overcome the limitations of soluble calcium reporters, we developed a genetically 62 encoded probe designed to detect microtubule-proximal increases in [Ca 2+ ] i . This probe, 63 dubbed TubeCamp, comprises the calcium-sensitive derivative of GFP, GCamp3 11 , 64 fused with the microtubule-binding domain of ensconsin (EMTB) 12 . GCamp 65 fluorescence emission increases upon calcium binding 11 while fusions of EMTB with 66 fluorescent proteins have been used in mammalian 13 , amphibian 14, 15 and invertebrate 14 67 cells to label microtubules. To determine whether TubeCamp reports on microtubule-68 proximal elevated [Ca 2+ ] i , TubeCamp and R-Geco, a calcium-sensitive reporter protein 16 69were expressed in Xenopus oocytes which were then wounded to elicit a local [Ca 2+ ] i 70 increase and microtubule reorganization 17 . Before wounding, the oocyte cortex 71

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“…Surprisingly, it is nonetheless quite accurate (Liu, Shao, Wang, & Liu, 2014). Most recently, they have provided evidence that meiotic progression may depend on nanodomain calcium increases in the region around the meiotic spindle (Li, Leblanc, He, & Liu, 2016). …”

Section: Introductionmentioning

confidence: 99%

Living Xenopus oocytes, eggs, and embryos as models for cell division

Varjabedian

Kita

2018

Mitosis and Meiosis Part A

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Xenopus laevis has long been a popular model for studies of development and, based on the use of cell-free extracts derived from its eggs, as a model for reconstitution of cell cycle regulation and other basic cellular processes. However, work over the last several years has shown that intact Xenopus eggs and embryos are also powerful models for visualization and characterization of cell cycle-regulated cytoskeletal dynamics. These findings were something of a surprise, given that the relatively low opacity of Xenopus eggs and embryos was assumed to make them poor subjects for live-cell imaging. In fact, however, the high tolerance for light exposure, the development of new imaging approaches, new probes for cytoskeletal components and cytoskeletal regulators, and the ease of microinjection make the Xenopus oocytes, eggs, and embryos one of the most useful live-cell imaging models among the vertebrates. In this review, we describe the basics of using X. laevis as a model organism for studying cell division and outline experimental approaches for imaging cytoskeletal components in vivo in X. laevis embryos and eggs.

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Spindle function inXenopusoocytes involves possible nanodomain calcium signaling

Cited by 16 publications

References 61 publications

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

Calcium nanodomains in spindles

Living Xenopus oocytes, eggs, and embryos as models for cell division

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