Heritable changes in division speed accompany the diversification of single T cell fate

Rapid clonal expansion of antigen specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naive T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging "T cell family" has never been measured with single-cell resolution. Here, we utilize continuous live-cell imaging in vitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies and model-based computational analysis using a novel Bayesian inference framework for tree-structured data reveals a segregation into heritably fast and slow dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective re-expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases the latter of which coincides with specification of memory vs. effector fate.

Recognition and Repair of Oxidatively Generated DNA Lesions in Plasmid DNA by a Facilitated Diffusion Mechanism

The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of base excision repair (BER) pathway initiated by the bifunctional DNA glycosylase NEIL1. In this work we reported that the excision kinetics of the single Sp lesions site-specifically embedded in the covalently closed circular DNA plasmids (contour length 2686 base pairs) by NEIL1 are biphasic under single-turnover conditions ([NEIL1]>>[SpDNApl]) in contrast to monophasic excision kinetics of the same lesions embedded in147-mer Sp-modified DNA duplexes. Under conditions of a large excess of plasmid DNA base pairs over NEIL1 molecules, the kinetics of excision of Sp lesions are biphasic in nature, exhibiting an initial burst phase, followed by a slower rate of formation of excision products The burst phase is associated with NEIL1-DNA plasmid complexes, while the slow kinetic phase is attributed to the dissociation of non-specific NEIL1-DNA complexes. The amplitude of the burst phase is limited in amplitude because of the competing non-specific binding of NEIL1 to unmodified DNA sequences flanking the lesion. A numerical analysis of the incision kinetics yielded a value of φ » 0.03 for the fraction of NEIL1 encounters with plasmid molecules that result in the excision of the Sp lesion, and a characteristic dissociation time of non-specific NEIL1-DNA complexes (τ-ns » 8 s). The estimated average DNA translocation distance of NEIL1 is ~80 base pairs. This estimate suggests that facilitated diffusion enhances the probability that NEIL1 can locate its substrate embedded in an excess of unmodified plasmid DNA nucleotides by a factor of ~ 10.

Pursuit predation with intermittent locomotion in zebrafish

The control of a predator's locomotion is critical to its ability to capture prey. Flying animals adjust their heading continuously with control similar to guided missiles. However, many animals do not move with rapid continuous motion, but rather interrupt their progress with frequent pauses. To understand how such intermittent locomotion may be controlled during predation, we examined the kinematics of zebrafish (Danio rerio) as they pursued larval prey of the same species. Like many fishes, zebrafish move with discrete burst-and-coast swimming. We found that the change in heading and tail excursion during the burst phase was linearly related to the prey's bearing. These results suggest a strategy, which we call intermittent pure pursuit, that offers advantages in sensing and control. This control strategy is similar to perception and path-planning algorithms required in the design of some autonomous robots and may be common to a diversity of animals.

Estonian geminate plosives: some durational characteristics

Consonants in Estonian occur in three quantity degrees: short, long, and overlong. Plosives have also been described as lenis and fortis. Long and fortis correspond to geminate. As single plosives have been described acoustically several times, only overall durations of geminates have been reported. The present study examines durational patterns of geminate plosives in Estonian and their relations to the same patterns in single plosives. Influences of articulation place, quantity, syllable structure, and speech style on overall duration, burst duration, and voiced transition were sought. The biggest differences in duration occurred due to quantity degree. Some influence of syllable structure occurred, similar to previous findings. Patterns were mostly similar to those in singleton plosives, although there were some differences in voicing. Almost no difference compared to singletons with respect to burst phase duration was found. Kokkuvõte. Liis Ermus: Eesti keele geminaatklusiilid: kestust puudutavaid tunnuseid. Eesti keele konsonandid esinevad kolmes vältes: lühike, pikk ja ülipikk. Sulghäälikuid on kirjeldatud ka leenise ja fortisena. Pikk ja ülipikk välde ning fortis vastavad geminaadile. Lühikeste sulghäälikute akustikat eesti keeles on korduvalt uuritud, kuid geminaatide kohta on esitatud vaid mõningast kestusinfot. Käesolev uurimus keskendus eesti keele geminaatsulghäälikute kestuses esinevatele korrapäradele ja nende võrdlusele lühikeste sulghäälikute kestusmustritega. Vaadeldi häälduskoha, välte, silbistruktuuri ja kõnestiili mõju häälikute üldkestusele, samuti vallandumisfaasi ja helilise algussiirde kestusele. Suurimad kestuserinevused olid tingitud välteerinevustest. Ilmnes mõningane silbistruktuuri mõju, mis oli kooskõlas eelnevate uurimustega. Kestusmustrid olid üldjoontes sarnased lühikeste sulghäälikute kestusmustritega, põhilised erinevused esinesid helilisuses. Vallandumisfaasi kestuses seevastu polnud võrreldes lühikeste häälikutega peaaegu mingeid erinevusi. Märksõnad: eesti keel, geminaadid, sulghäälikud, kestus, seotud kõne, akustiline foneetika

Insights into the dynamic control of breathing revealed through cell-type-specific responses to substance P

The rhythm generating network for breathing must continuously adjust to changing metabolic and behavioral demands. Here, we examined network-based mechanisms in the mouse preBötzinger complex using substance P, a potent excitatory modulator of breathing frequency and stability, as a tool to dissect network properties that underlie dynamic breathing. We find that substance P does not alter the balance of excitation and inhibition during breaths or the duration of the resulting refractory period. Instead, mechanisms of recurrent excitation between breaths are enhanced such that the rate that excitation percolates through the network is increased. We propose a conceptual framework in which three distinct phases of inspiration, the burst phase, refractory phase, and percolation phase, can be differentially modulated to control breathing dynamics and stability. Unraveling mechanisms that support this dynamic control may improve our understanding of nervous system disorders that destabilize breathing, many of which involve changes in brainstem neuromodulatory systems.

Increasing vitrification temperature improves the cryo-electron microscopy reconstruction

At the initial stage of the cryo-electron microcopy (cryo-EM) samples irradiated by electrons, the cryo-EM samples suffer from a rapid “burst” phase (first 3~4 e−/Å2) of beam induced motion (BIM) which is too fast to be corrected by existing motion correction software, and lowers the quality of the initial frames. Therefore, these least radiation damaged, but ruined frames are commonly excluded or down-weighted during data processing, which reduces the undamaged signals in the reconstruction and decreases the reconstruction resolution by weakening the averaging power. Here, we show that increasing the freezing temperature of cryo-EM samples suppresses the BIM in this phase. The quality of initial frames is partially recovered after BIM correction and is better than that of subsequent frames in certain frames. Incorporating these initial frames into the reconstruction increases the resolution, at an equivalent of ~60% extra data. Moreover, these frames are least radiation damaged, thus preserves the high quality cryo-EM density of radiation sensitive residues. Such density is usually damaged or very weak in the canonical 3D reconstruction. In addition, we found that a different kind of radiation damage neglected previously occurs in the per-frame reconstruction after the exposure of 2.5 e−/Å2. Such radiation damage distorts the density of atoms. The deformation can be avoided by only including the frames from the first 2.5 e−/Å2 into the reconstruction. Overall, the high temperature freezing not only provides extra undamaged signal to the reconstruction, but also increases the resolution of the reconstruction.

Short-term synaptic dynamics control the activity phase of neurons in an oscillatory network

In oscillatory systems, neuronal activity phase is often independent of network frequency. Such phase maintenance requires adjustment of synaptic input with network frequency, a relationship that we explored using the crab, Cancer borealis, pyloric network. The burst phase of pyloric neurons is relatively constant despite a > two fold variation in network frequency. We used noise input to characterize how input shape influences burst delay of a pyloric neuron, and then used dynamic clamp to examine how burst phase depends on the period, amplitude, duration, and shape of rhythmic synaptic input. Phase constancy across a range of periods required a proportional increase of synaptic duration with period. However, phase maintenance was also promoted by an increase of amplitude and peak phase of synaptic input with period. Mathematical analysis shows how short-term synaptic plasticity can coordinately change amplitude and peak phase to maximize the range of periods over which phase constancy is achieved.

Short-Term Synaptic Dynamics Control the Activity Phase of Neurons in an Oscillatory Network

In oscillatory systems, neuronal activity phase is often independent of network frequency. Such phase maintenance requires adjustment of synaptic input with network frequency, a relationship that we explored using the crab, Cancer borealis, pyloric network. The burst phase of pyloric neurons is relatively constant despite a >2-fold variation in network frequency. We used noise input to characterize how input shape influences burst delay of a pyloric neuron, and then used dynamic clamp to examine how burst phase depends on the period, amplitude, duration, and shape of rhythmic synaptic input. Phase constancy across a range of periods required a proportional increase of synaptic duration with period. However, phase maintenance was also promoted by an increase of amplitude and peak phase of synaptic input with period. Mathematical analysis shows how short-term synaptic plasticity can coordinately change amplitude and peak phase to maximize the range of periods over which phase constancy is achieved.