Biology subjects

Lehmann, L.

Publications and source records attributed to Lehmann, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Interferon-induced activation state of circulating dendritic cells and monocytes triggered by yellow fever vaccination correlates with early protective antibody responses

Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14 and 28 days after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on day 3 and 7 after vaccination as well as cell type specific responses and temporal dynamics. Single cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses.

immunology↗

bioRxiv2024

Functional assessment of a kcnb1 knock-out zebrafish to model KCNB1-related neurodevelopmental and epileptic disorders

KEY POINTSO_LIkcnb1 is expressed in distinct cell subtypes and various regions of the central nervous system in zebrafish C_LIO_LIBrain anatomy and neuronal circuits are not disrupted in the kcnb1 loss-of-function zebrafish model C_LIO_LILoss of kcnb1 leads to altered behavior phenotype, light and sound-induced locomotor impairments C_LIO_LIkcnb1 knock-out zebrafish exhibit increased locomotor sensitivity to PTZ and elevated expression of epileptogenesis-related genes C_LIO_LIkcnb1-/- larvae show spontaneous and provoked epileptiform-like electrographic activity associated with disrupted GABA regulation C_LI ObjectiveKCNB1 encodes an -subunit of the delayed-rectifier voltage-dependent potassium channel Kv2.1. De novo pathogenic variants of KCNB1 have been linked to developmental and epileptic encephalopathies (DEE), diagnosed in early childhood and sharing limited treatment options. Loss-of-function (LOF) of KCNB1 with dominant negative effects has been proposed as the pathogenic mechanism in these disorders. Here, we aim to characterize a knock-out (KO) zebrafish line targeting kcnb1 (kcnb1+/- and kcnb1-/-) for investigating DEEs. MethodsThis study presents the phenotypic analysis of a kcnb1 knock-out zebrafish model, obtained by CRISPR/Cas9 mutagenesis. Through a combination of immunohistochemistry, behavioral assays, electrophysiological recordings, and neurotransmitter quantifications, we have characterized the expression, function, and impact of this kcnb1 LOF model at early stages of development. ResultsIn wild-type larval zebrafish, kcnb1 was found in various regions of the central nervous system and in diverse cell subtypes including neurons, oligodendrocytes and microglial cells. Both kcnb1+/- and kcnb1-/- zebrafish displayed impaired swimming behavior and "epilepsy-like" features that persisted through embryonic and larval development, with variable severity. When exposed to the chemoconvulsant pentylenetetrazol (PTZ), both mutant models showed elevated locomotor activity. In addition, PTZ-exposed kcnb1-/- larvae exhibited higher bdnf mRNA expression and activated c-Fos positive neurons in the telencephalon. This same model presents spontaneous and provoked epileptiform-like electrographic activity associated with disrupted GABA regulation. In this KO model, neuronal circuit organization remained unaffected. SignificanceWe conclude that kcnb1 knock-out in zebrafish leads to early-onset phenotypic features reminiscent of DEEs, affecting neuronal functions and primarily inhibitory pathways in developing embryonic and larval brains. This study highlights the relevance of this model for investigating developmental neuronal signaling pathways in KCNB1-related DEEs.

neuroscience↗

bioRxiv2024

The coevolution of learning schedules and teaching enhances cumulative knowledge and drives a teacher-innovator syndrome

Natural selection shapes how individuals learn and acquire knowledge from their environment. Under the right conditions, this can lead to the evolution of learning schedules--how individuals allocate resources to acquire knowledge throughout their lifespan--that promote the accumulation of knowledge across generations ("cumulative knowledge" or "cumulative culture"). In spite of having been observed across multiple taxa, the role of parental teaching in this evolutionary process remains understudied. Using mathematical modelling, we show that learning schedules and parental teaching coevolve, resulting in greater time spent learning individually and innovating, as well as greater inter-generational transfer of knowledge from parent to offspring. These outcomes together enhance cumulative knowledge. Our analyses further reveal that within populations, selection typically favours an association between teaching and individual learning whereby some individuals innovate and teach within the family ("knowledge producers" with extensive knowledge), while others teach less and learn socially outside of the family ("knowledge scroungers" with less knowledge). Overall, our findings indicate that the coevolution of learning schedules and teaching promotes knowledge accumulation within and between generations, and favours diversity within and between populations in knowledge acquisition, possession and transmission.

evolutionary biology↗

bioRxiv2024

Central place foragers, prey depletion halos, and how behavioral niche partitioning promotes consumer coexistence

Many seabirds congregate in large colonies for breeding, a time when they are central place foragers. An influential idea in seabird ecology posits that competition during breeding results in an area of reduced prey availability around colonies, a phenomenon known as Ashmoles halo, and that this limits colony size. This idea has gained empirical support, including the finding that species coexisting within a colony might be able to do so by foraging on a single prey species but at different distances. Here, we provide a comprehensive mathematical model for central place foragers exploiting a single prey in a two-dimensional environment, where the prey distribution is the result of intrinsic birth and death, movement in space and mortality due to foraging birds (we also consider a variant tailored toward colonial social insects). Bird predation at different distances occurs according to an ideal free foraging distribution that maximizes prey delivery under flight and search costs. We fully characterize the birds ideal free distribution and the prey distribution it generates. Our results show that prey depletion halos around breeding colonies are a robust phenomenon and that the birds ideal free distribution is sensitive to prey movement. Furthermore, coexistence of several seabird species on a single prey easily emerges through behavioral niche partitioning whenever trait differences between species entail trade-offs between efficiently exploiting a scarce prey close to the colony and a more abundant prey far away. Such behavioral-based coexistence-inducing mechanism should generalize to other habitat and diet choice scenarios. Significance statementThis study presents a mathematical model to explore the distribution of foraging trips among seabirds breeding on isolated islands, providing insight into the emergence of prey depletion halos around colonies. Our findings reveal that such halos are a robust feature of central place foraging, independent of prey dynamics. Additionally, the model shows that trait-mediated niche partitioning promotes coexistence among species through behavioral segregation into different circular zones around the island. This partitioning occurs despite a shared preference to forage close to the island, where flight costs are lowest. The study advances understanding of ecological coexistence mechanisms and suggests broader applicability to other predator-prey systems beyond seabird ecology, offering a new perspective on community assembly under shared preferences.

ecology↗

bioRxiv2024

Machine Learning and Directed Evolution of Base Editing Enzymes

As we enter the era of CRISPR medicines, base editors (BEs) emerged as one of the most promising tools to treat genetic associated diseases. However, unintended bystander editing beyond the target nucleotide poses a challenge to their translation into effective therapies. While many efforts have been made in the design of a universal enzyme with minimal bystander editing, the context dependent activity represents a major challenge for base editing-based therapies. In this work, we designed a sequence-specific guide RNA library with 3-extensions and detected guides that were able to reduce bystander and increase editing efficiency in a context dependent manner. The best candidate was later used for phage assisted non-continuous evolution to find a new generation of precise base editors. Simultaneously, we use protein language models trained on massive protein sequence datasets to find the evolutionarily plausible mutational patterns that can improve deaminase activity and precision. Both strategies provide a collection of precise TadA variants that not only drastically reduced bystander edits, but also was not in detriment of on-target activity. Our findings introduce a guide/enzyme parallel engineering pipeline, which lays the foundation for the development of new personalized genome editing strategies, ultimately enhancing the safety and precision of this groundbreaking technology.

synthetic biology↗

bioRxiv2023

Evolution of environmentally mediated social interactions under isolation by distance

Many social interactions happen indirectly via modifications of the environment, e.g. through the secretion of functional compounds or the depletion of renewable resources. Here, we derive the selection gradient on a quantitative trait affecting dynamical environmental variables that feed back on reproduction and survival in a finite patch-structured population subject to isolation by distance. Our analysis shows that the selection gradient depends on how a focal individual influences the fitness of all future individuals in the population through modifications of the environmental variables they experience, weighted by the neutral relatedness between recipients and the focal. The evolutionarily relevant trait-driven environmental modifications are formalized as the extended phenotypic effects of an individual, quantifying how a trait change in an individual in the present affects the environmental variables in all patches at all future times. When the trait affects reproduction and survival through a payoff function, the selection gradient can be expressed in terms of extended phenotypic effects weighted by scaled-relatedness. We show how to compute extended phenotypic effects, relatedness, and scaled-relatedness using Fourier analysis, which allow us to investigate a broad class of environmentally mediated social interactions in a tractable way. We use our approach to study the evolution of a trait controlling the costly production of some lasting commons (e.g. a common-pool resource or a toxic compound) that can diffuse in space and persist in time. We show that indiscriminate posthumous spite readily evolves in this scenario. More generally whether selection favours environmentally mediated altruism or spite is determined by the spatial correlation between an individuals lineage and the commons originating from its patch. The sign of this correlation depends on interactions between dispersal patterns and the commons renewal dynamics. More broadly, we suggest that selection can favour a wide range of social behaviours when these have carry-over effects in space and time.

evolutionary biology↗

bioRxiv2023

Evolution of semi-Kantian preferences in two-player assortative interactions with complete and incomplete information and plasticity

We model the evolution of preferences guiding behavior in pairwise interactions in group-structured populations. The model uses long-term evolution theory to examine different interaction scenarios, including conditional preference expression upon recognition of the partners type. We apply the model to the evolution of semi-Kantian preferences at the fitness level, which combine self-interest and a Kantian interest evaluating own behavior in terms of consequences for own fitness if the partner also adopted this behavior. We seek the convergence stable and uninvadable value of the Kantian coefficient, i.e., the weight attached to the Kantian interest, a quantitative trait varying between zero and one. We consider three scenarios: (a) incomplete information; (b) complete information and incomplete plasticity; and (c) complete information and complete plasticity, where individuals not only recognize the type of their interaction partner (complete information), but also conditionally express the Kantian coefficient upon it (complete plasticity). For (a), the Kantian coefficient generally evolves to equal the coefficient of neutral relatedness between interacting individuals; for (b), it evolves to a value that depends on demographic and interaction assumptions, while for (c) there are generally multiple uninvadable types, including the type whereby an individual is a pure Kantian when interacting with individuals of the same type and applies the Kantian coefficient that is uninvadable under complete information with zero relatedness when interacting with a different typed individual. Overall, our model connects several concepts for analysing the evolution of behavior rules for strategic interactions that have been emphasized in different and sometimes isolated literatures.

evolutionary biology↗

bioRxiv2023

Prior flavivirus immunity skews the yellow fever vaccine response to expand cross-reactive antibodies with increased risk of antibody dependent enhancement of Zika and dengue virus infection

Human pathogenic flaviviruses pose a significant health concern and vaccination is the most effective instrument to control their circulation. How pre-existing immunity to antigenically related viruses modulates immunization outcome remains poorly understood. In this study, we evaluated the effect of vaccination against tick-borne encephalitis virus (TBEV) on the epitope immunodominance and immunogenicity of the yellow fever 17D vaccine (YF17D) in a cohort of 250 human vaccinees. Following YF17D vaccination, all study participants seroconverted and generated protective neutralizing antibody titers. At day 28, TBEV pre-immunity did not affect the polyclonal neutralizing response which largely depended on the IgM fraction. We found that sera from TBEV-immunized individuals enhanced YF17D vaccine virus infection via antibody-dependent enhancement (ADE). Upon vaccination, individuals with TBEV pre-immunity had higher concentrations of cross-reactive IgG antibodies with limited neutralizing capacity against YF17D whereas vaccinees without prior flavivirus exposure showed a non-cross-reacting response. Using a set of recombinant YF17D envelope protein mutants displaying different epitopes, we identified quaternary epitopes as the primary target of neutralizing antibodies. Sequential immunizations redirected the IgG response towards the pan-flavivirus fusion loop epitope (FLE) with the potential to mediate enhancement of dengue and Zika virus infections whereas TBEV naive individuals elicited an IgG response directed towards neutralizing epitopes without an enhancing effect. We propose that the YF17D vaccine effectively conceals the FLE and primes a neutralizing IgG response in individuals with no prior flavivirus exposure. In contrast, the response in TBEV-experienced recipients favors weakly-neutralizing, cross-reactive epitopes potentially increasing the risk of severe dengue and Zika disease due to ADE.

immunology↗

bioRxiv2022

Foraging for locally and spatially varying resources: Where exploitation competition, local adaptation and kin selection meet

In patch- or habitat-structured populations different processes can lead to diversity at different scales. While spatial heterogeneity generates spatially disruptive selection favoring variation between patches, local competition can lead to locally disruptive selection promoting variation within patches. So far, almost all theory has studied these two processes in isolation. Here, we use mathematical modelling to investigate how resource variation within and between habitats influences the evolution of variation in a consumer population where individuals compete in finite patches connected by dispersal. We find that locally and spatially disruptive selection typically act in concert, favoring polymorphism under a significantly wider range of conditions than when in isolation. But when patches are small and dispersal between them is low, kin competition inhibits the emergence of polymorphism, especially when driven by local competition. We further use our model to clarify what comparisons between trait and neutral genetic differentiation (Qst/Fst comparisons) can tell about the nature of selection. Overall, our results help understand the interaction between two major drivers of diversity: locally and spatially disruptive selection; and how this interaction is modulated by the unavoidable effects of kin selection under limited dispersal.

evolutionary biology↗

bioRxiv2022

On Hamilton's rule, the evolution of behavior rules and the wizardry of control theory

This paper formalizes selection on a quantitative trait affecting the evolution of behavior (or development) rules through which individuals act and react with their surroundings. Combining Hamiltons marginal rule for selection on scalar traits and concepts from optimal control theory, a necessary first-order condition for the evolutionary stability of the trait in a group-structured population is derived. The model, which is of intermediate level of complexity, fills a gap between the formalization of selection on evolving traits that are directly conceived as actions (no phenotypic plasticity) and selection on evolving traits that are conceived as strategies or function valued actions (complete phenotypic plasticity). By conceptualizing individuals as open deterministic dynamical systems expressing incomplete phenotypic plasticity, the model captures selection on a large class of phenotypic expression mechanisms, including developmental pathways and learning under life-history trade-offs. As an illustration of the results, a first-order condition for the evolutionary stability of behavior response rules from the social evolution literature is re-derived, strengthened, and generalized. All results of the paper also generalize directly to selection on multidimensional quantitative traits affecting behavior rule evolution, thereby covering neural and gene network evolution.

evolutionary biology↗

bioRxiv2022

On the interpretation of the operation of natural selection in class-structured populations

Biological adaptation is the outcome of allele-frequency change by natural selection. At the same time, populations are usually class structured as individuals occupy different states such as age, sex, or stage. This is known to result in the differential transmission of alleles through non-heritable fitness differences called class transmission, also affecting allele-frequency change even in the absence of selection. How does one then isolate the allele-frequency change due to selection from that owing to class transmission? We decompose one-generational allele-frequency change in terms of effects of selection and class transmission, and show how reproductive values can be used to reach a decomposition between any two distant generations of the evolutionary process. This provides a missing relationship between multigenerational allele-frequency change and the operation of selection. It also allows to define a measure of fitness summarizing the effect of selection in a multigenerational evolutionary process, which connects asymptotically to invasion fitness.

evolutionary biology↗

bioRxiv2022

Life-history and deleterious mutation rate coevolution

The cost of germline maintenance gives rise to a trade-off between lowering the deleterious mutation rate and investing in life history functions. Therefore, life history and the mutation rate coevolve, but this coevolution is not well understood. We develop a mathematical model to analyse the evolution of resource allocation traits, which simultaneously affect life history and the deleterious mutation rate. First, we show that the invasion fitness of such resource allocation traits can be approximated by the basic reproductive number of the least-loaded class; the expected lifetime production of offspring without deleterious mutations born to individuals without deleterious mutations. Second, we apply the model to investigate (i) the coevolution of reproductive effort and germline maintenance and (ii) the coevolution of age-at-maturity and germline maintenance. This analysis provides two resource allocation predictions when exposure to environmental mutagens is higher. First, selection favours higher allocation to germline maintenance, even if it comes at the expense of life history functions, and leads to a shift in allocation towards reproduction rather than survival. Second, life histories tend to be faster, characterized by individuals with shorter lifespans and smaller body sizes at maturity. Our results suggest that mutation accumulation via the cost of germline maintenance can be a major force shaping life-history traits.

evolutionary biology↗

bioRxiv2021

Evolution of warfare by resource raiding favors polymorphism in belligerence and bravery

From protists to primates, intergroup aggression and warfare over resources has been observed in several taxa whose populations typically consist of groups connected by limited genetic mixing. Here, we model the co-evolution between four traits relevant to this setting: (i) investment into common-pool resource production within groups ("helping"); (ii) proclivity to raid other groups to appropriate their resources ("bel-ligerence"); and investments into (iii) defense and (iv) offense of group contests ("defensive and offensive bravery"). We show that when traits co-evolve, the population often experiences disruptive selection favouring two morphs: "Hawks", who express high levels of both belligerence and offensive bravery; and "Doves", who express neither. This social polymorphism involves further among-traits associations when the fitness costs of helping and bravery interact. In particular if helping is antagonistic with both forms of bravery, co-evolution leads to the coexistence of individuals that either: (i) do not participate into common-pool resource production but only in its defense and appropriation ("Scrounger Hawks"); or (ii) only invest into common pool resource production ("Producer Doves"). Provided groups are not randomly mixed, these findings are robust to several modelling assumptions. This suggests that inter-group aggression is a potent mechanism in favoring within-group social diversity and behavioural syndromes.

evolutionary biology↗

bioRxiv2021

Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome

The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and reveal that ALC1 preferentially slides nucleosomes away from DNA breaks.

biophysics↗

bioRxiv2021

Metacommunities, fitness and gradual evolution

We analyze the evolution of a multidimensional quantitative trait in a class structured focal species interacting with other species in a wider metacommunity. The evolutionary dynamics in the focal species as well as the ecological dynamics of the whole metacommunity is described as a continuous time process with birth, physiological development, dispersal, and death given as rates that can depend on the state of the whole metacommunity. This can accommodate complex local community and global metacommunity environmental feedbacks owing to inter- and intra-specific interactions, as well as local environmental stochastic fluctuations. For the focal species, we derive a fitness measure for a mutant allele affecting class-specific trait expression. Using classical results from geometric singular perturbation theory, we provide a detailed proof that if the effect of the mutation on phenotypic expression is small ("weak selection"), the large system of dynamical equations needed to describe selection on the mutant allele in the metacommunity can be reduced to a single ordinary differential equation on the arithmetic mean mutant allele frequency that is of constant sign. This invariance on allele frequency entails the mutant either dies out or will out-compete the ancestral resident (or wild) type. Moreover, the directional selection coefficient driving arithmetic mean allele frequency can be expressed as an inclusive fitness effect calculated from the resident metacommunity alone, and depends, as expected, on individual fitness differentials, relatedness, and reproductive values. This formalizes the Darwinian process of gradual evolution driven by random mutation and natural selection in spatially and physiologically class structured metacommunities.

evolutionary biology↗

bioRxiv2020

Hamilton's rule, gradual evolution, and the optimal (feedback) control of reaction norms and other function-valued traits

Most traits expressed by organisms, such as gene expression profiles, developmental trajectories, behavioural sequences and reaction norms are function-valued traits (colloquially "phenotypically plastic traits"), since they vary across an individuals age and in response to various internal and/or external factors (state variables). Furthermore, most organisms live in populations subject to limited genetic mixing and are thus likely to interact with their relatives. We here formalise selection on genetically determined function-valued traits of individuals interacting in a group-structured population, by deriving the marginal version of Hamiltons rule for function-valued traits. This rule simultaneously gives a condition for the invasion of an initially rare mutant function-valued trait and its ultimate fixation in the population (invasion thus implies substitution). Hamiltons rule thus underlies the gradual evolution of function-valued traits and gives rise to necessary first-order conditions for their uninvadability (evolutionary stability). We develop a novel analysis using optimal control theory and differential game theory, to simultaneously characterise and compare the first-order conditions of (i) open-loop traits - functions of time (or age) only, and (ii) closed-loop (state-feedback) traits - functions of both time and state variables. We show that closed-loop traits can be represented as the simpler open-loop traits when individuals do no interact or when they interact with clonal relatives. Our analysis delineates the role of state-dependence and interdependence between individuals for trait evolution, which has implications to both life-history theory and social evolution.

evolutionary biology↗

bioRxiv2020

The components of directional and disruptive selection in heterogeneous group-structured populations

We derive how directional and disruptive selection operate on scalar traits in a heterogeneous group-structured population for a general class of models. In particular, we assume that each group in the population can be in one of a finite number of states, where states can affect group size and/or other environmental variables, at a given time. Using up to second-order perturbation expansions of the invasion fitness of a mutant allele, we derive expressions for the directional and disruptive selection coefficients, which are sufficient to classify the singular strategies of adaptive dynamics. These expressions include first- and second-order perturbations of individual fitness (expected number of settled offspring produced by an individual, possibly including self through survival); the first-order perturbation of the stationary distribution of mutants (derived here explicitly for the first time); the first-order perturbation of pairwise relatedness; and reproductive values, pairwise and three-way relatedness, and stationary distribution of mutants, each evaluated under neutrality. We introduce the concept of individual k-fitness (defined as the expected number of settled offspring of an individual for which k - 1 randomly chosen neighbors are lineage members) and show its usefulness for calculating relatedness and its perturbation. We then demonstrate that the directional and disruptive selection coefficients can be expressed in terms individual k-fitnesses with k = 1, 2, 3 only. This representation has two important benefits. First, it allows for a significant reduction in the dimensions of the system of equations describing the mutant dynamics that needs to be solved to evaluate explicitly the two selection coefficients. Second, it leads to a biologically meaningful interpretation of their components. As an application of our methodology, we analyze directional and disruptive selection in a lottery model with either hard or soft selection and show that many previous results about selection in group-structured populations can be reproduced as special cases of our model.

evolutionary biology↗

bioRxiv2020

Mechanism of DNA surface exploration and operator bypassing during target search

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 s. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

biophysics↗