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1.
J Cell Biol ; 223(9)2024 Sep 02.
Article in English | MEDLINE | ID: mdl-38913324

ABSTRACT

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.


Subject(s)
Actin Cytoskeleton , Actins , Animals, Genetically Modified , Green Fluorescent Proteins , Quail , Animals , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Actins/metabolism , Actins/genetics , Actin Cytoskeleton/metabolism , Morphogenesis , Embryo, Nonmammalian/metabolism
2.
Dev Cell ; 59(6): 705-722.e8, 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38354738

ABSTRACT

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.


Subject(s)
Myocytes, Cardiac , Wnt Signaling Pathway , ortho-Aminobenzoates , Myocytes, Cardiac/metabolism , Cell Differentiation/genetics , Cell Lineage/genetics , Wnt Signaling Pathway/genetics , Mesoderm
3.
Nat Commun ; 14(1): 3101, 2023 05 29.
Article in English | MEDLINE | ID: mdl-37248263

ABSTRACT

During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity.


Subject(s)
Actins , Adaptor Proteins, Signal Transducing , Humans , Animals , Mice , Adaptor Proteins, Signal Transducing/metabolism , Nuclear Lamina/metabolism , Cell Cycle Proteins , YAP-Signaling Proteins , Blastocyst/metabolism , Lamins
4.
Front Cell Dev Biol ; 10: 864522, 2022.
Article in English | MEDLINE | ID: mdl-35676934

ABSTRACT

Mechanical forces are now recognized as key cellular effectors that together with genetic and cellular signals physically shape and pattern tissues and organs during development. Increasing efforts are aimed toward understanding the less explored role of mechanical forces in controlling cell fate decisions in embryonic development. Here we discuss recent examples of how differential forces feedback into cell fate specification and tissue patterning. In particular, we focus on the role of actomyosin-contractile force generation and transduction in affecting tissue morphogenesis and cell fate regulation in the embryo.

5.
Nature ; 585(7825): 404-409, 2020 09.
Article in English | MEDLINE | ID: mdl-32848249

ABSTRACT

To implant in the uterus, the mammalian embryo first specifies two cell lineages: the pluripotent inner cell mass that forms the fetus, and the outer trophectoderm layer that forms the placenta1. In many organisms, asymmetrically inherited fate determinants drive lineage specification2, but this is not thought to be the case during early mammalian development. Here we show that intermediate filaments assembled by keratins function as asymmetrically inherited fate determinants in the mammalian embryo. Unlike F-actin or microtubules, keratins are the first major components of the cytoskeleton that display prominent cell-to-cell variability, triggered by heterogeneities in the BAF chromatin-remodelling complex. Live-embryo imaging shows that keratins become asymmetrically inherited by outer daughter cells during cell division, where they stabilize the cortex to promote apical polarization and YAP-dependent expression of CDX2, thereby specifying the first trophectoderm cells of the embryo. Together, our data reveal a mechanism by which cell-to-cell heterogeneities that appear before the segregation of the trophectoderm and the inner cell mass influence lineage fate, via differential keratin regulation, and identify an early function for intermediate filaments in development.


Subject(s)
Cell Lineage , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Keratins/metabolism , Actins/metabolism , Animals , Cell Division , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/metabolism , Ectoderm/cytology , Embryo, Mammalian/embryology , Female , Humans , Intermediate Filaments/metabolism , Mice , Microtubules/metabolism , Multiprotein Complexes/metabolism , Trophoblasts/cytology
6.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118572, 2020 01.
Article in English | MEDLINE | ID: mdl-31678117

ABSTRACT

Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100 ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.


Subject(s)
Kinesins/metabolism , Microtubules/physiology , Organelles/metabolism , Animals , Biological Transport , Cells, Cultured , Drosophila , Fluorescence , Kinetics , Microtubules/metabolism , Spectrometry, Fluorescence
7.
J Mol Biol ; 431(6): 1148-1159, 2019 03 15.
Article in English | MEDLINE | ID: mdl-30790630

ABSTRACT

Chromatin remodeling is fundamental for the dynamical changes in transcriptional programs that occur during development and stem cell differentiation. The histone acetyltransferase Kat6b is relevant for neurogenesis in mouse embryos, and mutations of this gene cause intellectual disability in humans. However, the molecular mechanisms involved in Kat6b mutant phenotype and the role of this chromatin modifier in embryonic stem (ES) cells remain elusive. In this work, we show that Kat6b is expressed in ES cells and is repressed during differentiation. Moreover, we found that this gene is regulated by the pluripotency transcription factors Nanog and Oct4. To study the functional relevance of Kat6b in ES cells, we generated a Kat6b knockout ES cell line (K6b-/-) using CRISPR/Cas9. Fluorescence correlation spectroscopy analyses suggest a more compact chromatin organization in K6b-/- cells and impaired interactions of Oct4 and Nanog with chromatin. Remarkably, K6b-/- cells showed a reduced efficiency to differentiate to neural lineage. These results reveal a role of Kat6b as a modulator of chromatin plasticity, its impact on chromatin-transcription factors interactions and its influence on cell fate decisions during neural development.


Subject(s)
Chromatin/metabolism , Embryonic Stem Cells/cytology , Histone Acetyltransferases/metabolism , Nanog Homeobox Protein/metabolism , Neurogenesis , Octamer Transcription Factor-3/metabolism , Animals , CRISPR-Cas Systems , Embryonic Stem Cells/metabolism , Gene Expression Regulation , Histone Acetyltransferases/genetics , Male , Mice, Nude
8.
Mech Dev ; 154: 60-63, 2018 12.
Article in English | MEDLINE | ID: mdl-29753812

ABSTRACT

Pluripotent stem cells (PSCs) are capable of self-renewing and producing all cell types derived from the three germ layers in response to developmental cues, constituting an important promise for regenerative medicine. Pluripotency depends on specific transcription factors (TFs) that induce genes required to preserve the undifferentiated state and repress other genes related to differentiation. The transcription machinery and regulatory components such as TFs are recruited dynamically on their target genes making it essential exploring their dynamics in living cells to understand the transcriptional output. Non-invasive and very sensitive fluorescence microscopy methods are making it possible visualizing the dynamics of TFs in living specimens, complementing the information extracted from studies in fixed specimens and bulk assays. In this work, we briefly describe the basis of these microscopy methods and review how they contributed to our knowledge of the function of TFs relevant to embryo development and cell differentiation in a variety of systems ranging from single cells to whole organisms.


Subject(s)
Embryonic Development/physiology , Transcription Factors/metabolism , Animals , Cell Differentiation/physiology , Germ Layers/embryology , Germ Layers/metabolism , Humans , Microscopy, Fluorescence/methods , Pluripotent Stem Cells/enzymology , Pluripotent Stem Cells/metabolism , Transcription, Genetic/physiology
9.
Cell ; 173(3): 776-791.e17, 2018 04 19.
Article in English | MEDLINE | ID: mdl-29576449

ABSTRACT

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.


Subject(s)
Actins/chemistry , Blastocyst/metabolism , Microtubules/metabolism , Myosin Type II/chemistry , Animals , Cell Communication , Cytoskeletal Proteins/chemistry , Embryo, Mammalian , Embryonic Development , Female , Green Fluorescent Proteins , Imaging, Three-Dimensional , Mice , Mice, Inbred C57BL , Morula , RNA, Small Interfering/metabolism , Tight Junctions
10.
Curr Top Dev Biol ; 128: 37-58, 2018.
Article in English | MEDLINE | ID: mdl-29477170

ABSTRACT

The early mouse embryo offers a phenomenal system to dissect how changes in the mechanisms controlling cell fate are integrated with morphogenetic events at the single-cell level. New technologies based on live imaging have enabled the discovery of dynamic changes in the regulation of single genes, transcription factors, and epigenetic mechanisms directing early cell fate decision in the early embryo. Here, we review recent progress in linking molecular dynamic events occurring at the level of the single cell in vivo, to some of the key morphogenetic changes regulating early mouse development.


Subject(s)
Blastocyst/cytology , Cell Lineage , Embryonic Development , Mammals/embryology , Animals , Blastocyst/metabolism , Gene Expression Regulation, Developmental , Humans , Transcription Factors/metabolism
11.
Nat Protoc ; 12(7): 1458-1471, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28686586

ABSTRACT

Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.


Subject(s)
DNA/metabolism , Single-Cell Analysis/methods , Spectrometry, Fluorescence/methods , Transcription Factors/metabolism , Animals , Embryo, Mammalian , Mice , Protein Binding , Time Factors
12.
Front Cell Neurosci ; 10: 184, 2016.
Article in English | MEDLINE | ID: mdl-27507935

ABSTRACT

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 ± 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 ± 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies.

13.
Cell ; 165(1): 75-87, 2016 Mar 24.
Article in English | MEDLINE | ID: mdl-27015308

ABSTRACT

Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos. In blastocysts, the TFs Oct4 and Sox2, which control pluripotency, bind DNA more stably in pluripotent than in extraembryonic cells. By contrast, in the four-cell embryo, Sox2 engages in more long-lived interactions than does Oct4. Sox2 long-lived binding varies between blastomeres and is regulated by H3R26 methylation. Live-cell tracking demonstrates that those blastomeres with more long-lived binding contribute more pluripotent progeny, and reducing H3R26 methylation decreases long-lived binding, Sox2 target expression, and pluripotent cell numbers. Therefore, Sox2-DNA binding predicts mammalian cell fate as early as the four-cell stage. More generally, we reveal the dynamic repartitioning of TFs between DNA sites driven by physiological epigenetic changes. VIDEO ABSTRACT.


Subject(s)
SOXB1 Transcription Factors/metabolism , Animals , Blastocyst/metabolism , CARD Signaling Adaptor Proteins/metabolism , DNA/metabolism , Diffusion , Down-Regulation , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/analysis , Histones/metabolism , Kinetics , Methylation , Mice , Octamer Transcription Factor-3/metabolism , Spectrometry, Fluorescence
14.
Dev Cell ; 34(4): 435-47, 2015 Aug 24.
Article in English | MEDLINE | ID: mdl-26279486

ABSTRACT

Every cell in our body originates from the pluripotent inner mass of the embryo, yet it is unknown how biomechanical forces allocate inner cells in vivo. Here we discover subcellular heterogeneities in tensile forces, generated by actomyosin cortical networks, which drive apical constriction to position the first inner cells of living mouse embryos. Myosin II accumulates specifically around constricting cells, and its disruption dysregulates constriction and cell fate. Laser ablations of actomyosin networks reveal that constricting cells have higher cortical tension, generate tension anisotropies and morphological changes in adjacent regions of neighboring cells, and require their neighbors to coordinate their own changes in shape. Thus, tensile forces determine the first spatial segregation of cells during mammalian development. We propose that, unlike more cohesive tissues, the early embryo dissipates tensile forces required by constricting cells via their neighbors, thereby allowing confined cell repositioning without jeopardizing global architecture.


Subject(s)
Blastocyst Inner Cell Mass/cytology , Blastocyst Inner Cell Mass/physiology , Animals , Biomechanical Phenomena , Cadherins/metabolism , Cell Adhesion , Cell Count , Cell Lineage , Down-Regulation , Female , Humans , Mice, Inbred C57BL , Myosin Type II/metabolism , Subcellular Fractions/metabolism
15.
Nano Lett ; 13(12): 6156-63, 2013.
Article in English | MEDLINE | ID: mdl-24219503

ABSTRACT

α-synuclein (AS) is a small (140 amino acids), abundant presynaptic protein, which lacks a unique secondary structure in aqueous solution. Amyloid aggregates of AS in dopaminergic neurons of the midbrain are the hallmark of Parkinson's disease (PD). The process of aggregation involves a series of complex structural transitions from innocuous monomeric AS to oligomeric, presumably neurotoxic, forms and finally to fibril formation. Despite its potential importance for understanding PD pathobiology and devising rational, targeted therapeutic strategies, the details of the aggregation process remain largely unknown. Methodologies and reagents capable of controlling the aggregation kinetics are essential tools for the investigation of the molecular mechanisms of amyloid diseases. In this work, we investigated the influence of citrate-capped gold nanoparticles on the aggregation kinetics of AS using a fluorescent probe (MFC) sensitive to the polarity of the molecular microenvironment via excited state intramolecular proton transfer (ESIPT). The particular effects on the half time, nucleation time, and growth rate were ascertained. Gold nanoparticles produced a strong acceleration of protein aggregation with an influence on both the nucleation and growth phases of the overall mechanism. The effects were dependent on the size and concentration of the nanoparticles, being strongest for nanoparticles 10 nm in diameter, which produced a 3-fold increase in the overall aggregation rate at concentrations as low as 20 nM.


Subject(s)
Amyloid/chemistry , Gold/chemistry , Parkinson Disease/pathology , alpha-Synuclein/chemistry , Humans , Kinetics , Metal Nanoparticles/chemistry , Parkinson Disease/etiology , Protein Structure, Secondary
16.
PLoS One ; 8(1): e54846, 2013.
Article in English | MEDLINE | ID: mdl-23382986

ABSTRACT

It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+) channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+) current. Accordingly, in the present work we found that the Ca(2+) current flowing through P/Q-type Ca(2+) channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca(2+) current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K(+) stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca(2+) channels.


Subject(s)
Calcium Channels, P-Type/metabolism , Calcium Channels, Q-Type/metabolism , Chromaffin Cells/metabolism , Secretory Vesicles/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Exocytosis/physiology , Mice
17.
J Neurochem ; 116(2): 155-63, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21073467

ABSTRACT

In neuroendocrine cells, such as adrenal chromaffin cells, the exocytosis of hormone-filled vesicles is triggered by a localized Ca(2+) increase that develops after the activation of voltage-dependent Ca(2+) channels. To reach the fusion competent state, vesicles have to go through a series of maturation steps that involve the detachment from cytoskeletal proteins, docking and priming. However, the fusion readiness of vesicles will also depend on their proximity to the calcium source. The immediately releasable pool is a small group of ready-to-fuse vesicles, whose fusion is tightly coupled to Ca(2+) entry through channels. Recent work indicates that such coupling is not produced by a random distribution between vesicles and channels, but would be the result of a specific interaction of immediately releasable vesicles with particular Ca(2+) channel subtypes. The immediately releasable pool is able to sustain, with high efficiency, the secretion triggered by the small and localized Ca(2+) gradients produced by brief depolarizations at low frequencies, like action potentials at basal conditions in adrenal chromaffin cells.


Subject(s)
Chromaffin Cells/metabolism , Exocytosis/physiology , Neuroendocrine Cells/metabolism , Synaptic Vesicles/metabolism , Animals , Calcium Channels/metabolism , Humans , Neuroendocrine Cells/cytology , Time Factors
18.
Cell Calcium ; 43(2): 155-64, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17561253

ABSTRACT

Chromaffin cell exocytosis is triggered by Ca(2+) entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca(2+) channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31+/-3 fF for control conditions (I(Ca): 25+/-2 pA/pF). The N-type channel blocker omega-conotoxin-GVIA affected neither I(Ca) nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker omega-Agatoxin-IVA decreased I(Ca) by 37% but strongly reduced the immediately releasable pool (upper limit: 6+/-1 fF). We used alpha1A subunit knockout mice to corroborate that P/Q Ca(2+) channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6+/-1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50 mM K(+)) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells.


Subject(s)
Calcium Channels, P-Type/physiology , Calcium Channels, Q-Type/physiology , Calcium Channels/metabolism , Chromaffin Cells/metabolism , Exocytosis/physiology , Animals , Calcium Channel Blockers/pharmacology , Chromaffin Cells/drug effects , Electric Capacitance , Electric Stimulation , Mice , Mice, Knockout , Patch-Clamp Techniques
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