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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.

Assuntos

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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.

Assuntos

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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.

Assuntos

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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.

RESUMO

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.

Assuntos

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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.

Assuntos

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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.

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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.

Assuntos