Mechanisms of glioblastoma replication: Ca2+ flares and Cl- currents.

Glioblastoma (GBM) is amongst the deadliest types of cancers, with no resolutive cure currently available. GBM cell proliferation in the patient's brain is a complex phenomenon controlled by multiple mechanisms. The aim of this study was to determine whether the ionic fluxes controlling cell duplication could represent a target for GBM therapy. In this work, we combined multi-channel Ca2+ and Cl- imaging, optical tweezers, electrophysiology and immunohistochemistry to describe the role of ion fluxes in mediating the cell volume changes that accompany mitosis of U87 GBM cells. We identified three main steps: (i) in round GBM cells undergoing mitosis, during the transition from anaphase to telophase and cytokinesis, large Ca2+ flares occur, reaching values of 0.5-1 µM; (ii) these Ca2+ flares activate Ca2+-dependent Cl- channels, allowing the entry of Cl- ions; (iii) to maintain osmotic balance, GBM cells swell to complete mitosis. This sequence of steps was validated by electrophysiological experiments showing that Cl- channels are activated either directly or indirectly by Ca2+, and by additional live-cell imaging experiments. Cl- channel blockers with different molecular structures, such as niflumic acid and carbenoxolone, blocked GBM replication by arresting GBM cells in a round configuration. These results describe the central role of Ca2+ flares and Cl- fluxes during mitosis and show that inhibition of Ca2+-activated Cl- channels blocks GBM replication, opening the way to new approaches for the clinical treatment of GBM. Implications: Our work identifies ionic fluxes occurring during cell division as targets for devising novel therapies for the glioblastoma treatment.

Unfolding and identification of membrane proteins in situ.

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.

The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony.

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.

Mechanotransduction in hippocampal neurons operates under localized low picoNewton forces.

There is growing evidence suggesting that mechanical properties of CNS neurons may play an important regulatory role in cellular processes. Here, we employ an oscillatory optical tweezers (OOT) to exert a local indentation with forces in the range of 5-50 pN. We found that single local indentation above a threshold of 13 ± 1 pN evokes a transient intracellular calcium change, whereas repeated mechanical stimulations induce a more sustained and variable calcium response. Importantly, neurons were able to differentiate the magnitude of mechanical stimuli. Chemical perturbation and whole-cell patch clamp recordings suggest that mechanically evoked response requires the influx of extracellular calcium through transmembrane ion channels. Moreover, we observed a mechanically evoked activation of the CAMKII and small G protein RhoA. These results all together suggest that mechanical signaling among developed neurons fully operates in neuronal networks under physiological conditions.

Calcium flares and compartmentalization in rod photoreceptors.

Rod photoreceptors are composed of a soma and an inner segment (IS) connected to an outer segment (OS) by a thin cilium. OSs are composed of a stack of ∼800 lipid discs surrounded by the plasma membrane where phototransduction takes place. Intracellular calcium plays a major role in phototransduction and is more concentrated in the discs, where it can be incorporated and released. To study calcium dynamics in rods, we used the fluorescent calcium dye CaSiR-1 AM working in the near-infrared (NIR) (excitation at 650 and emission at 664 nm), an advantage over previously used dyes. In this way, we investigated calcium dynamics with an unprecedented accuracy and most importantly in semidark-adapted conditions. We observed light-induced drops in [Ca2+]i with kinetics similar to that of photoresponses recorded electrophysiologically. We show three properties of the rods. First, intracellular calcium and key proteins have concentrations that vary from the OS base to tip. At the OS base, [Ca2+]i is ∼80 nM and increases up to ∼200 nM at the OS tip. Second, there are spontaneous calcium flares in healthy and functional rod OSs; these flares are highly localized and are more pronounced at the OS tip. Third, a bright flash of light at 488 nm induces a drop in [Ca2+]i at the OS base but often a flare at the OS tip. Therefore, rod OSs are not homogenous structures but have a structural and functional gradient, which is a fundamental aspect of transduction in vertebrate photoreceptors.

Mechanosensitivity is an essential component of phototransduction in vertebrate rods.

Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation-of the order of 10 pN-applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods' phototransduction.

Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation.

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration.