Microscopic interactions control a structural transition in active mixtures of microtubules and molecular motors.

Microtubules and molecular motors are essential components of the cellular cytoskeleton, driving fundamental processes in vivo, including chromosome segregation and cargo transport. When reconstituted in vitro, these cytoskeletal proteins serve as energy-consuming building blocks to study the self-organization of active matter. Cytoskeletal active gels display rich emergent dynamics, including extensile flows, locally contractile asters, and bulk contraction. However, it is unclear how the protein-protein interaction kinetics set their contractile or extensile nature. Here, we explore the origin of the transition from extensile bundles to contractile asters in a minimal reconstituted system composed of stabilized microtubules, depletant, adenosine 5'-triphosphate (ATP), and clusters of kinesin-1 motors. We show that the microtubule-binding and unbinding kinetics of highly processive motor clusters set their ability to end-accumulate, which can drive polarity sorting of the microtubules and aster formation. We further demonstrate that the microscopic time scale of end-accumulation sets the emergent time scale of aster formation. Finally, we show that biochemical regulation is insufficient to fully explain the transition as generic aligning interactions through depletion, cross-linking, or excluded volume interactions can drive bundle formation despite end-accumulating motors. The extensile-to-contractile transition is well captured by a simple self-assembly model where nematic and polar aligning interactions compete to form either bundles or asters. Starting from a five-dimensional organization phase space, we identify a single control parameter given by the ratio of the different component concentrations that dictates the material-scale organization. Overall, this work shows that the interplay of biochemical and mechanical tuning at the microscopic level controls the robust self-organization of active cytoskeletal materials.

Dissipation and energy propagation across scales in an active cytoskeletal material.

Living systems are intrinsically nonequilibrium: They use metabolically derived chemical energy to power their emergent dynamics and self-organization. A crucial driver of these dynamics is the cellular cytoskeleton, a defining example of an active material where the energy injected by molecular motors cascades across length scales, allowing the material to break the constraints of thermodynamic equilibrium and display emergent nonequilibrium dynamics only possible due to the constant influx of energy. Notwithstanding recent experimental advances in the use of local probes to quantify entropy production and the breaking of detailed balance, little is known about the energetics of active materials or how energy propagates from the molecular to emergent length scales. Here, we use a recently developed picowatt calorimeter to experimentally measure the energetics of an active microtubule gel that displays emergent large-scale flows. We find that only approximately one-billionth of the system's total energy consumption contributes to these emergent flows. We develop a chemical kinetics model that quantitatively captures how the system's total thermal dissipation varies with ATP and microtubule concentrations but that breaks down at high motor concentration, signaling an interference between motors. Finally, we estimate how energy losses accumulate across scales. Taken together, these results highlight energetic efficiency as a key consideration for the engineering of active materials and are a powerful step toward developing a nonequilibrium thermodynamics of living systems.

Odd dynamics of living chiral crystals.

Active crystals are highly ordered structures that emerge from the self-organization of motile objects, and have been widely studied in synthetic1,2 and bacterial3,4 active matter. Whether persistent  crystalline order can emerge  in groups of autonomously developing multicellular organisms is currently unknown. Here we show that swimming starfish embryos spontaneously assemble into chiral crystals that span thousands of spinning organisms and persist for tens of hours. Combining experiments, theory and simulations, we demonstrate that the formation, dynamics and dissolution of these living crystals are controlled by the hydrodynamic properties and the natural development of embryos. Remarkably, living chiral crystals exhibit self-sustained chiral oscillations as well as various unconventional deformation response behaviours recently predicted for odd elastic materials5,6. Our results provide direct experimental evidence for how non-reciprocal interactions between autonomous multicellular components may facilitate non-equilibrium phases of chiral active matter.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells.

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

A Micromachined Picocalorimeter Sensor for Liquid Samples with Application to Chemical Reactions and Biochemistry.

Calorimetry has long been used to probe the physical state of a system by measuring the heat exchanged with the environment as a result of chemical reactions or phase transitions. Application of calorimetry to microscale biological samples, however, is hampered by insufficient sensitivity and the difficulty of handling liquid samples at this scale. Here, a micromachined calorimeter sensor that is capable of resolving picowatt levels of power is described. The sensor consists of low-noise thermopiles on a thin silicon nitride membrane that allow direct differential temperature measurements between a sample and four coplanar references, which significantly reduces thermal drift. The partial pressure of water in the ambient around the sample is maintained at saturation level using a small hydrogel-lined enclosure. The materials used in the sensor and its geometry are optimized to minimize the noise equivalent power generated by the sensor in response to the temperature field that develops around a typical sample. The experimental response of the sensor is characterized as a function of thermopile dimensions and sample volume, and its capability is demonstrated by measuring the heat dissipated during an enzymatically catalyzed biochemical reaction in a microliter-sized liquid droplet. The sensor offers particular promise for quantitative measurements on biological systems.

Measuring the Energetic Costs of Embryonic Development.

What are the thermodynamic costs of development? In this issue of Developmental Cell, Rodenfels et al. (2019) demonstrate that the high energetic cost of coordinated cell division that is regulated by phospho-signaling gives rise to a measurable periodicity in the heat dissipated during zebrafish embryogenesis.

Self-straining of actively crosslinked microtubule networks.

Cytoskeletal networks are foundational examples of active matter and central to self-organized structures in the cell. In vivo, these networks are active and densely crosslinked. Relating their large-scale dynamics to the properties of their constituents remains an unsolved problem. Here, we study an in vitro active gel made from aligned microtubules and XCTK2 kinesin motors. Using photobleaching, we demonstrate that the gel's aligned microtubules, driven by motors, continually slide past each other at a speed independent of the local microtubule polarity and motor concentration. This phenomenon is also observed, and remains unexplained, in spindles. We derive a general framework for coarse graining microtubule gels crosslinked by molecular motors from microscopic considerations. Using microtubule-microtubule coupling through a force-velocity relationship for kinesin, this theory naturally explains the experimental results: motors generate an active strain rate in regions of changing polarity, which allows microtubules of opposite polarities to slide past each other without stressing the material.

From cytoskeletal assemblies to living materials.

Many subcellular structures contain large numbers of cytoskeletal filaments. Such assemblies underlie much of cell division, motility, signaling, metabolism, and growth. Thus, understanding cell biology requires understanding the properties of networks of cytoskeletal filaments. While there are well established disciplines in biology dedicated to studying isolated proteins - their structure (Structural Biology) and behaviors (Biochemistry) - it is much less clear how to investigate, or even just describe, the structure and behaviors of collections of cytoskeletal filaments. One approach is to use methodologies from Mechanics and Soft Condensed Matter Physics, which have been phenomenally successful in the domains where they have been traditionally applied. From this perspective, collections of cytoskeletal filaments are viewed as materials, albeit very complex, 'active' materials, composed of molecules which use chemical energy to perform mechanical work. A major challenge is to relate these material level properties to the behaviors of the molecular constituents. Here we discuss this materials perspective and review recent work bridging molecular and network scale properties of the cytoskeleton, focusing on the organization of microtubules by dynein as an illustrative example.

Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge.

Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser-like manner across a protein bridge that connects the inner and outer membranes.

Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization.

Cytoplasmic dynein-1 is a minus-end-directed motor protein that transports cargo over long distances and organizes the intracellular microtubule (MT) network. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, resulting in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, suggesting that minus-end accumulations of motors produce network-scale contractile stresses. Our data provide a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the cytoskeleton, for example, during mitotic spindle assembly.

Bridging length scales to measure polymer assembly.

Time-resolvable quantitative measurements of polymer concentration are very useful to elucidate protein polymerization pathways. There are numerous techniques to measure polymer concentrations in purified protein solutions, but few are applicable in vivo. Here we develop a methodology combining microscopy and spectroscopy to overcome the limitations of both approaches for measuring polymer concentration in cells and cell extracts. This technique is based on quantifying the relationship between microscopy and spectroscopy measurements at many locations. We apply this methodology to measure microtubule assembly in tissue culture cells and Xenopus egg extracts using two-photon microscopy with FLIM measurements of FRET. We find that the relationship between FRET and two-photon intensity quantitatively agrees with predictions. Furthermore, FRET and intensity measurements change as expected with changes in acquisition time, labeling ratios, and polymer concentration. Taken together, these results demonstrate that this approach can quantitatively measure microtubule assembly in complex environments. This methodology should be broadly useful for studying microtubule nucleation and assembly pathways of other polymers.

Developing and Testing a Bayesian Analysis of Fluorescence Lifetime Measurements.

FRET measurements can provide dynamic spatial information on length scales smaller than the diffraction limit of light. Several methods exist to measure FRET between fluorophores, including Fluorescence Lifetime Imaging Microscopy (FLIM), which relies on the reduction of fluorescence lifetime when a fluorophore is undergoing FRET. FLIM measurements take the form of histograms of photon arrival times, containing contributions from a mixed population of fluorophores both undergoing and not undergoing FRET, with the measured distribution being a mixture of exponentials of different lifetimes. Here, we present an analysis method based on Bayesian inference that rigorously takes into account several experimental complications. We test the precision and accuracy of our analysis on controlled experimental data and verify that we can faithfully extract model parameters, both in the low-photon and low-fraction regimes.

Active contraction of microtubule networks.

Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction.

Concentration of various trace elements in the rat retina and their distribution in different structures.

Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the total amount of trace elements in retina from adult male Sprague-Dawley rats (n = 6). Concentration of trace elements within individual retinal areas in frozen sections of the fellow eye was established with the use of two methodologies: (1) particle-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) and (2) synchrotron X-ray fluorescence (SXRF) microscopy. The most abundant metal in the retina was zinc, followed by iron and copper. Nickel, manganese, chromium, cobalt, selenium and cadmium were present in very small amounts. The PIXE and SXRF analysis yielded a non-homogenous pattern distribution of metals in the retina. Relatively high levels of zinc were found in the inner part of the photoreceptor inner segments (RIS)/outer limiting membrane (OLM), inner nuclear layer and plexiform layers. Iron was found to accumulate in the retinal pigment epithelium/choroid layer and RIS/OLM. Copper in turn, was localised primarily in the RIS/OLM and plexiform layers. The trace elements iron, copper, and zinc exist in different amounts and locations in the rat retina.

Prevalence of short stature in Saudi children and adolescents.

BACKGROUND AND OBJECTIVE: Data on stature in Saudi children and adolescents are limited. The objective of this report was to establish the national prevalence of short stature in Saudi children and adolescents. DESIGN AND SETTING: Community-based, cross-sectional study conducted over 2 years (2004, 2005) PATIENTS AND METHODS: The national data set of the Saudi reference was used to calculate the stature for age for children and adolescents 5 to 18 years of age. Using the 2007 World Health Organization (WHO) reference, the prevalence of moderate and severe short stature was defined as the proportion of children whose standard deviation score for stature for age was less than -2 and -3, respectively. In addition, the 2000 Center for Disease Control (CDC) and the older 1978 National Center for Health Statistics (NCHS)/WHO references were used for comparison. RESULTS: Using the 2007 WHO reference, sample size in the Saudi reference was 19 372 healthy children and adolescents 5 to 17 years of age, with 50.8% being boys. The overall prevalence of moderate and severe short stature in boys was 11.3% and 1.8%, respectively; and in girls, 10.5% and 1.2%, respectively. The prevalence of moderate short stature was 12.1%, 11% and 11.3% in boys and 10.9%, 11.3% and 10.5% in girls when the 1978 WHO, the 2000 CDC and the 2007 WHO references were used, respectively. CONCLUSIONS: The national prevalence of short stature in Saudi children and adolescents is intermediate compared with the international level. Improvement in the socioeconomic and health status of children and adolescents should lead to a reduction in the prevalence of short stature.

Prevalence of malnutrition in Saudi children: a community-based study.

BACKGROUND AND OBJECTIVE: There is no published information on the prevalence of malnutrition in Saudi Arabia. The objective of this study was to establish the prevalence data. METHODS: The prevalence of nutritional indicators in the form of underweight, stunting, and wasting in a national sample of children younger than 5 years of age was calculated using the new WHO standards as reference. Calculations were performed using the corresponding WHO software. The prevalence of moderate and severe underweight, wasting and stunting, was defined as the proportion of children whose weight for age, weight for height, and height for age were below -2 and -3 standard deviation scores, respectively. RESULTS: The number of children younger than 5 years of age was 15 516 and 50.5% were boys. The prevalence of moderate and severe underweight was 6.9% and 1.3%, respectively. The prevalence of moderate and severe wasting was 9.8% and 2.9%, respectively. Finally, the prevalence of moderate and severe stunting was 10.9% and 2.8%, respectively. The prevalence was lower in girls for all indicators. Comparison of the prevalence of nutritional indicators in selected countries demonstrates large disparity with an intermediate position for Saudi Arabia. CONCLUSION: This report establishes the national prevalence of malnutrition among Saudi children. Compared to data from other countries, these prevalence rates are still higher than other countries with less economic resources, indicating that more efforts are needed to improve the nutritional status of children.

Regional disparity in prevalence of malnutrition in Saudi children.

OBJECTIVE: To evaluate the regional difference in the prevalence of malnutrition in Saudi children. METHODS: Data for this study were collected over 2 years (2004 and 2005). A cross-sectional representative sample of the Saudi population of healthy children below 5 years of age was used to calculate the prevalence of malnutrition. The study was carried out in the College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia. Body measurements of the weight, length, and height were performed according to standard recommendations. Standard deviation scores were determined using the Lambda, Mu, and Sigma (LMS) statistical methodology. The 1978 NCHS/WHO growth reference was used for the calculation of prevalence of underweight, wasting, and stunting defined as the proportion of children whose weight for age, weight for height, and height for age was below minus standard deviation (-2 SD) for Northern, Southwestern, and Central regions of the Kingdom of Saudi Arabia. Chi-square test was used to assess the difference in prevalence between regions, and a p<0.05 was considered significant. RESULTS: The sample size of children <5 years of age in Central region was 5067, Southwestern 2285, and Northern 2933. The prevalence of underweight was 4%, 19.7% and 5.5%, that of wasting was 6.5%, 16.7% and 6.5% and of stunting was 6.4%, 13.2% and 6.4% in the Central, Southwestern, and Northern regions indicating a significantly-higher prevalence in Southwestern compared to other regions (p<0.001). CONCLUSION: This report revealed a high prevalence of significant nutritional indicators in the Southwestern regions than in other regions. This finding indicates that this region should be given priority for further studies to identify causes, and to design health promotion programs.

Prevalence of overweight and obesity in Saudi children and adolescents.

BACKGROUND AND OBJECTIVE: There is limited information on overweight and obesity in Saudi children and adolescents. The objective of this study was to establish the national prevalence of overweight and obesity in Saudi children and adolescents. METHODS: The 2005 Saudi reference data set was used to calculate the body mass index (BMI) for children aged 5 to 18 years. Using the 2007 WHO reference, the prevalence of overweight, obesity and severe obesity were defined as the proportion of children with a BMI standard deviation score more than +1, +2 and +3, respectively. The 2000 CDC reference was also used for comparison. RESULTS: There were 19 317 healthy children and adolescents from 5 to 18 years of age, 50.8% of whom were boys. The overall prevalence of overweight, obesity and severe obesity in all age groups was 23.1%, 9.3% and 2%, respectively. A significantly lower prevalence of overweight (23.8 vs 20.4; P<.001) and obesity (9.5 vs 5.7; P<.001) was found when the CDC reference was used. CONCLUSIONS: This report establishes baseline national prevalence rates for overweight, obesity and severe obesity in Saudi children and adolescents, indicating intermediate levels between developing and industrialized countries. Measures should be implemented to prevent further increases in the numbers of overweight school-age children and adolescents and the associated health hazards.

Pattern of sex differences in growth of Saudi children and adolescents.

BACKGROUND: Although variations in growth between boys and girls have been reported, detailed descriptions according to age and growth parameters are not available. OBJECTIVE: The goal of this study was to determine the pattern and magnitude of differences in growth between boys and girls according to age that justify separate growth charts. METHODS: The data set was based on a cross-sectional representative sample of the Saudi population of healthy children and adolescents from birth to 19 years of age. Body measurements (length, height, weight, and head circumference) were performed according to standard recommendations; body mass index was also determined for each subject. The difference in growth between boys and girls was assessed based on z scores and percentiles (5th, 50th, and 95th) of growth parameters using 2 age groups (0-3 years and 2-19 years). The significance of the difference between boys and girls for any growth parameter was tested by ANCOVA. RESULTS: A total of 35,279 children and adolescents from birth to 19 years of age satisfied the criteria for growth measurements. There were 17,880 boys and 17,399 girls; all were Saudi nationals. The Saudi boys were generally taller and heavier than girls up to approximately 7 to 10 years of age. Thereafter, girls generally were taller and heavier than boys from 10 to 14-15 years of age. After that, boys again were taller and heavier. Similar variations were observed for body mass index and head circumference. The difference between boys and girls for each growth parameter was highly significant (P < 0.001). CONCLUSIONS: Based on analysis of these Saudi children and adolescents, the difference in growth between boys and girls was not uniform but depended on age. However, the pattern was remarkably consistent across all growth parameters and appears to reflect the timing of maturation between boys and girls.

Body mass index in Saudi Arabian children and adolescents: a national reference and comparison with international standards.

BACKGROUND AND OBJECTIVES: Because there are no reference standards for body mass index (BMI) in Saudi children, we established BMI reference percentiles for normal Saudi Arabian children and adolescents and compared them with international standards. SUBJECTS AND METHODS: Data from a stratified multistage probability sample were collected from the 13 health regions in Saudi Arabia, as part of a nationwide health profile survey of Saudi Arabian children and adolescents conducted to establish normal physical growth references. Selected households were visited by a trained team. Weight and length/height were measured and recorded following the WHO recommended procedures using the same equipment, which were subjected to both calibration and intra/interobserver variations. RESULTS: Survey of 11 874 eligible households yielded 35 275 full-term and healthy children and adolescents who were subjected to anthropometric measurements. Four BMI curves were produced, from birth to 36 months and 2 to 19 years for girls and boys. The 3rd , 5 th , 10th , 25th , 50th , 75th , 85th , 90th , 95th , and 97th percentiles were produced and compared with the WHO and CDC BMI charts. In the higher percentiles, the Saudi children differed from Western counterparts, indicating that Saudi children have equal or higher BMIs. CONCLUSION: The BMI curves reflect statistically representative BMI values for Saudi Arabian children and adolescents.