Sarcomere Disassembly and Transfection Efficiency in Proliferating Human iPSC-Derived Cardiomyocytes.

Contractility of the adult heart relates to the architectural degree of sarcomeres in individual cardiomyocytes (CMs) and appears to be inversely correlated with the ability to regenerate. In this study we utilized multiple imaging techniques to follow the sequence of sarcomere disassembly during mitosis resulting in cellular or nuclear division in a source of proliferating human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We observed that both mono- and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny. Within this source of highly proliferative hiPSC-CMs, treated with the CHIR99021 small molecule, we found that Wnt and Hippo signaling was more present when compared to metabolic matured non-proliferative hiPSC-CMs and adult human heart tissue. Furthermore, we found that CHIR99021 increased the efficiency of non-viral vector incorporation in high-proliferative hiPSC-CMs, in which fluorescent transgene expression became present after the chromosomal segregation (M phase). This study provides a tool for gene manipulation studies in hiPSC-CMs and engineered cardiac tissue. Moreover, our data illustrate that there is a complex biology behind the cellular and nuclear division of mono- and binuclear CMs, with a shared-phenomenon of sarcomere disassembly during mitosis.

FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State.

Cancer stem cells (CSCs) are located in dedicated niches, where they remain inert to chemotherapeutic drugs and drive metastasis. Although plasticity in the CSC pool is well appreciated, the molecular mechanisms implicated in the regulation of cancer stemness are still elusive. Here, we define a fucosylation-dependent reprogramming of colon cancer cells towards a stem cell-like phenotype and function. De novo transcriptional activation of Fut9 in the murine colon adenocarcinoma cell line, MC38, followed by RNA seq-based regulon analysis, revealed major gene regulatory networks related to stemness. Lewisx, Sox2, ALDH and CD44 expression, tumorsphere formation, resistance to 5-FU treatment and in vivo tumor growth were increased in FUT9-expressing MC38 cells compared to the control cells. Likewise, human CRC cell lines highly expressing FUT9 displayed phenotypic features of CSCs, which were significantly impaired upon FUT9 knock-out. Finally, in primary CRC FUT9+ tumor cells pathways related to cancer stemness were enriched, providing a clinically meaningful annotation of the complicity of FUT9 in stemness regulation and may open new avenues for therapeutic intervention.

Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling.

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

Nerve cells communicate using tiny electrical impulses called action potentials. Special proteins termed ion channels produce these electric signals by allowing specific charged particles, or ions, to pass in or out of cells across its membrane. When a nerve cell 'fires' an action potential, specific ion channels briefly open to let in a surge of positively charged ions which electrify the cell. Action potentials begin in the same place in each nerve cell, at an area called the axon initial segment. The large number of sodium channels at this site kick-start the influx of positively charged sodium ions ensuring that every action potential starts from the same place. Previous research has shown that, when action potentials begin, the concentration of calcium ions at the axon initial segment also increases, but it was not clear which ion channels were responsible for this entry of calcium. Channels that are selective for calcium ions are the prime candidates for this process. However, research in squid nerve cells gave rise to an unexpected idea by suggesting that sodium channels may not exclusively let in sodium but also allow some calcium ions to pass through. Hanemaaijer, Popovic et al. therefore wanted to test the routes that calcium ions take and see whether the sodium channels in mammalian nerve cells are also permeable to calcium. Experiments using fluorescent dyes to track the concentration of calcium in rat and human nerve cells showed that calcium ions accumulated at the axon initial segment when action potentials fired. Most of this increase in calcium could be stopped by treating the neurons with a toxin that prevents sodium channels from opening. Electrical manipulations of the cells revealed that, in this context, the calcium ions were effectively behaving like sodium ions. Human kidney cells were then engineered to produce the sodium channel protein. This confirmed that calcium and sodium ions were indeed both passing through the same channel. These results shed new light on the relationship between calcium ions and sodium channels within the mammalian nervous system and that this interplay occurs at the axon initial segment of the cell. Genetic mutations that 'nudge' sodium channels towards favoring calcium entry are also found in patients with autism spectrum disorders, and so this new finding may contribute to our understanding of these conditions.

Saltatory Conduction along Myelinated Axons Involves a Periaxonal Nanocircuit.

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.

High-Frequency Microdomain Ca2+ Transients and Waves during Early Myelin Internode Remodeling.

Ensheathment of axons by myelin is a highly complex and multi-cellular process. Cytosolic calcium (Ca2+) changes in the myelin sheath have been implicated in myelin synthesis, but the source of this Ca2+ and the role of neuronal activity is not well understood. Using one-photon Ca2+ imaging, we investigated myelin sheath formation in the mouse somatosensory cortex and found a high rate of spontaneous microdomain Ca2+ transients and large-amplitude Ca2+ waves propagating along the internode. The frequency of Ca2+ transients and waves rapidly declines with maturation and reactivates during remyelination. Unexpectedly, myelin microdomain Ca2+ transients occur independent of neuronal action potential generation or network activity but are nearly completely abolished when the mitochondrial permeability transition pores are blocked. These findings are supported by the discovery of mitochondria organelles in non-compacted myelin. Together, the results suggest that myelin microdomain Ca2+ signals are cell-autonomously driven by high activity of mitochondria during myelin remodeling.

A Versatile and Open-Source Rapid LED Switching System for One-Photon Imaging and Photo-Activation.

Combining fluorescence and transmitted light sources for microscopy is an invaluable method in cellular neuroscience to probe the molecular and cellular mechanisms of cells. This approach enables the targeted recording from fluorescent reporter protein expressing neurons or glial cells in brain slices and fluorescence-assisted electrophysiological recordings from subcellular structures. However, the existing tools to mix multiple light sources in one-photon microscopy are limited. Here, we present the development of several microcontroller devices that provide temporal and intensity control of light emitting diodes (LEDs) for computer controlled microscopy illumination. We interfaced one microcontroller with µManager for rapid and dynamic overlay of transmitted and fluorescent images. Moreover, on the basis of this illumination system we implemented an electronic circuit to combine two pulsed LED light sources for fast (up to 1 kHz) ratiometric calcium (Ca2+) imaging. This microcontroller enabled the calibration of intracellular Ca2+ concentration and furthermore the combination of Ca2+ imaging with optogenetic activation. The devices are based on affordable components and open-source hardware and software. Integration into existing bright-field microscope systems will take ∼1 day. The microcontroller based LED imaging substantially advances conventional illumination methods by limiting light exposure and adding versatility and speed.

The stochastic nature of action potential backpropagation in apical tuft dendrites.

In cortical pyramidal neurons, backpropagating action potentials (bAPs) supply Ca2+ to synaptic contacts on dendrites. To determine whether the efficacy of AP backpropagation into apical tuft dendrites is stable over time, we performed dendritic Ca2+ and voltage imaging in rat brain slices. We found that the amplitude of bAP-Ca2+ in apical tuft branches was unstable, given that it varied from trial to trial (termed "bAP-Ca2+ flickering"). Small perturbations in dendritic physiology, such as spontaneous synaptic inputs, channel inactivation, or temperature-induced changes in channel kinetics, can cause bAP flickering. In the tuft branches, the density of Na+ and K+ channels was sufficient to support local initiation of fast spikelets by glutamate iontophoresis. We quantified the time delay between the somatic AP burst and the peak of dendritic Ca2+ transient in the apical tuft, because this delay is important for induction of spike-timing dependent plasticity. Depending on the frequency of the somatic AP triplets, Ca2+ signals peaked in the apical tuft 20-50 ms after the 1st AP in the soma. Interestingly, at low frequency (<20 Hz), the Ca2+ peaked sooner than at high frequency, because only the 1st AP invaded tuft. Activation of dendritic voltage-gated Ca2+ channels is sensitive to the duration of the dendritic voltage transient. In apical tuft branches, small changes in the duration of bAP voltage waveforms cause disproportionately large increases in dendritic Ca2+ influx (bAP-Ca2+ flickering). The stochastic nature of bAP-Ca2+ adds a new perspective on the mechanisms by which pyramidal neurons combine inputs arriving at different cortical layers.NEW & NOTEWORTHY The bAP-Ca2+ signal amplitudes in some apical tuft branches randomly vary from moment to moment. In repetitive measurements, successful AP invasions are followed by complete failures. Passive spread of voltage from the apical trunk into the tuft occasionally reaches the threshold for local Na+ spike, resulting in stronger Ca2+ influx. During a burst of three somatic APs, the peak of dendritic Ca2+ in the apical tuft occurs with a delay of 20-50 ms depending on AP frequency.

Loss of Saltation and Presynaptic Action Potential Failure in Demyelinated Axons.

In cortical pyramidal neurons the presynaptic terminals controlling transmitter release are located along unmyelinated axon collaterals, far from the original action potential (AP) initiation site, the axon initial segment (AIS). Once initiated, APs will need to reliably propagate over long distances and regions of geometrical inhomogeneity like branch points (BPs) to rapidly depolarize the presynaptic terminals and confer temporally precise synaptic transmission. While axon pathologies such as demyelinating diseases are well established to impede the fidelity of AP propagation along internodes, to which extent myelin loss affects propagation along BPs and axon collaterals is not well understood. Here, using the cuprizone demyelination model, we performed optical voltage-sensitive dye (VSD) imaging from control and demyelinated layer 5 pyramidal neuron axons. In the main axon, we find that myelin loss switches the modality of AP propagation from rapid saltation towards a slow continuous wave. The duration of single AP waveforms at BPs or nodes was, however, only slightly briefer. In contrast, by using two-photon microscopy-guided loose-seal patch recordings from axon collaterals we revealed a presynaptic AP broadening in combination with a reduced velocity and frequency-dependent failure. Finally, internodal myelin loss was also associated with de novo sprouting of axon collaterals starting from the primary (demyelinated) axon. Thus, the loss of oligodendrocytes and myelin sheaths bears functional consequences beyond the main axon, impeding the temporal fidelity of presynaptic APs and affecting the functional and structural organization of synaptic connectivity within the neocortex.

Electrical behaviour of dendritic spines as revealed by voltage imaging.

Thousands of dendritic spines on individual neurons process information and mediate plasticity by generating electrical input signals using a sophisticated assembly of transmitter receptors and voltage-sensitive ion channel molecules. Our understanding, however, of the electrical behaviour of spines is limited because it has not been possible to record input signals from these structures with adequate sensitivity and spatiotemporal resolution. Current interpretation of indirect data and speculations based on theoretical considerations are inconclusive. Here we use an electrochromic voltage-sensitive dye which acts as a transmembrane optical voltmeter with a linear scale to directly monitor electrical signals from individual spines on thin basal dendrites. The results show that synapses on these spines are not electrically isolated by the spine neck to a significant extent. Electrically, they behave as if they are located directly on dendrites.

Imaging with organic indicators and high-speed charge-coupled device cameras in neurons: some applications where these classic techniques have advantages.

Dynamic calcium and voltage imaging is a major tool in modern cellular neuroscience. Since the beginning of their use over 40 years ago, there have been major improvements in indicators, microscopes, imaging systems, and computers. While cutting edge research has trended toward the use of genetically encoded calcium or voltage indicators, two-photon microscopes, and in vivo preparations, it is worth noting that some questions still may be best approached using more classical methodologies and preparations. In this review, we highlight a few examples in neurons where the combination of charge-coupled device (CCD) imaging and classical organic indicators has revealed information that has so far been more informative than results using the more modern systems. These experiments take advantage of the high frame rates, sensitivity, and spatial integration of the best CCD cameras. These cameras can respond to the faster kinetics of organic voltage and calcium indicators, which closely reflect the fast dynamics of the underlying cellular events.

Cortical dendritic spine heads are not electrically isolated by the spine neck from membrane potential signals in parent dendrites.

The evidence for an important hypothesis that cortical spine morphology might participate in modifying synaptic efficacy that underlies plasticity and possibly learning and memory mechanisms is inconclusive. Both theory and experiments suggest that the transfer of excitatory postsynaptic potential signals from spines to parent dendrites depends on the spine neck morphology and resistance. Furthermore, modeling of signal transfer in the opposite direction predicts that synapses on spine heads are not electrically isolated from voltages in the parent dendrite. In sharp contrast to this theoretical prediction, one of a very few measurements of electrical signals from spines reported that slow hyperpolarizing membrane potential changes are attenuated considerably by the spine neck as they spread from dendrites to synapses on spine heads. This result challenges our understanding of the electrical behavior of spines at a fundamental level. To re-examine the specific question of the transfer of dendritic signals to synapses of spines, we took advantage of a high-sensitivity Vm-imaging technique and carried out optical measurements of electrical signals from 4 groups of spines with different neck length and simultaneously from parent dendrites. The results show that spine neck does not filter membrane potential signals as they spread from the dendrites into the spine heads.

The spatio-temporal characteristics of action potential initiation in layer 5 pyramidal neurons: a voltage imaging study.

The spatial pattern of Na(+) channel clustering in the axon initial segment (AIS) plays a critical role in tuning neuronal computations, and changes in Na(+) channel distribution have been shown to mediate novel forms of neuronal plasticity in the axon. However, immunocytochemical data on channel distribution may not directly predict spatio-temporal characteristics of action potential initiation, and prior electrophysiological measures are either indirect (extracellular) or lack sufficient spatial resolution (intracellular) to directly characterize the spike trigger zone (TZ). We took advantage of a critical methodological improvement in the high sensitivity membrane potential imaging (V(m) imaging) technique to directly determine the location and length of the spike TZ as defined in functional terms. The results show that in mature axons of mouse cortical layer 5 pyramidal cells, action potentials initiate in a region ∼20 µm in length centred between 20 and 40 µm from the soma. From this region, the AP depolarizing wave invades initial nodes of Ranvier within a fraction of a millisecond and propagates in a saltatory fashion into axonal collaterals without failure at all physiologically relevant frequencies. We further demonstrate that, in contrast to the saltatory conduction in mature axons, AP propagation is non-saltatory (monotonic) in immature axons prior to myelination.

Effects of isoquinolonesulfonamides on action potential secretion coupling in pituitary cells.

BACKGROUND: Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high and steady prolactin release. Several intracellular proteins can mediate the action of calcium influx on prolactin secretion, including calmodulin-dependent protein kinases. Here, we studied effects of isoquinolonesulfonamides KN-62 and KN-93, calmodulin-dependent protein kinase inhibitors, and KN-92, an inactive analog, on spontaneous electrical activity, voltage-gated calcium influx, cyclic nucleotide production, and basal prolactin release. METHODS: The effects of these compounds on electrical activity and calcium signaling was measured in single lactotrophs and cyclic nucleotide production and prolactin release were determined in static culture and perifusion experiments of anterior pituitary cells from postpubertal female rats. RESULTS: KN-62 and KN-93 blocked basal prolactin release in a dose- and time-dependent manner, suggesting that calmodulin-dependent protein kinase could mediate the coupling of electrical activity and secretion. However, a similar effect on basal prolactin release was observed on application of KN-92, which does not inhibit this kinase. KN-93 also inhibited cAMP and cGMP production, but inhibition of prolactin release was independent of the status of cyclic nucleotide production. Single cell measurements revealed abolition of spontaneous and depolarization-induced electrical activity and calcium transients in KN-92/93-treated cells, with a time course comparable to that observed in secretory studies. CONCLUSIONS: The results suggest that caution should be used when interpreting data from studies using isoquinolonesulfonamides to evaluate the role of calmodulin-dependent protein kinases in excitable endocrine cells, because inactive compounds exhibit comparable effects on action potential secretion coupling to those of active compounds.

Dependence of STIM1/Orai1-mediated calcium entry on plasma membrane phosphoinositides.

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Multiple roles of Gi/o protein-coupled receptors in control of action potential secretion coupling in pituitary lactotrophs.

G(i/o) protein-coupled receptors, signaling through G protein-dependent and protein-independent pathways, have prominent effects on secretion by modulating calcium signaling and regulating the size of the releasable secretory pool, the rates of exocytosis and endocytosis, and de novo synthesis. Pituitary cells fire action potentials spontaneously, and the associated calcium influx is sufficient to maintain prolactin (PRL) release but not gonadotropin release at high and steady levels for many hours. Such secretion, termed intrinsic, spontaneous, or basal, reflects fusion of secretory vesicles triggered by the cell type-specific pattern of action potentials. In lactotrophs, activation of endothelin ET(A) and dopamine D(2) receptors causes inhibition of spontaneous electrical activity and basal adenylyl cyclase activity accompanied with inhibition of basal PRL release. Agonist-induced inhibition of cAMP production and firing of action potentials is abolished in cells with blocked pertussis toxin (PTX)-sensitive G(i/o) signaling pathway. However, agonist-induced inhibition of PRL release is only partially relieved in such treated cells, indicating that both receptors also inhibit exocytosis downstream of cAMP/calcium signaling. The PTX-insensitive step in agonist-induced inhibition of PRL release is not affected by inhibition of phosphoinositide 3-kinase and glycogen synthase kinase-3 but is partially rescued by downregulation of the G(z)alpha expression. Thus, ET(A) and D(2) receptors inhibit basal PRL release not only by blocking electrical activity but also by desensitizing calcium-secretion coupling.