Eukaryotic cell size regulation and its implications for cellular function and dysfunction.

Depending on cell type, environmental inputs, and disease, the cells in the human body can have widely different sizes. In recent years, it became clear that cell size is a major regulator of cell function. However, we are only beginning to understand how optimization of cell function determines a given cell's optimal size. Here, we review currently known size control strategies of eukaryotic cells, and the intricate link of cell size to intracellular biomolecular scaling, organelle homeostasis and cell cycle progression. We detail the cell size dependent regulation of early development and the impact of cell size on cell differentiation. Given the importance of cell size for normal cellular physiology, cell size control must account for changing environmental conditions. We describe how cells sense environmental stimuli, such as nutrient availability, and accordingly adapt their size by regulating cell growth and cell cycle progression. Moreover, we discuss the correlation of pathological states with misregulation of cell size, and how for a long time, this was considered a downstream consequence of cellular dysfunction. We review newer studies that reveal a reversed causality, with misregulated cell size leading to pathophysiological phenotypes such as senescence and aging. In summary, we highlight important roles of cell size in cellular function and dysfunction, which could have major implications for both diagnostics and treatment in the clinic.

Too big not to fail: Different paths lead to senescence of enlarged cells.

In this issue of Molecular Cell, Crozier et al.,1 Foy et al.,2 Manohar et al.,3 and Wilson et al.4 show how excessive cell growth caused by a temporary G1 arrest leads to permanent cell cycle exit at different stages of the cell cycle.

Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size.

Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to newborn cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each newborn cell regardless of cell size. Disrupting both Whi5 synthesis and chromatin-based partitioning weakens G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.

Segmentation, tracking and cell cycle analysis of live-cell imaging data with Cell-ACDC.

BACKGROUND: High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. RESULTS: We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae, histone Htb1 concentrations decrease with replicative age. CONCLUSIONS: Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis. Source code: https://github.com/SchmollerLab/Cell_ACDC.

Single-molecule experiments reveal the elbow as an essential folding guide in SMC coiled-coil arms.

Structural maintenance of chromosome (SMC) complexes form ring-like structures through exceptional elongated coiled-coils (CCs). Recent studies found that variable CC conformations, including open and collapsed forms, which might result from discontinuities in the CC, facilitate the diverse functions of SMCs in DNA organization. However, a detailed description of the SMC CC architecture is still missing. Here, we study the structural composition and mechanical properties of SMC proteins with optical tweezers unfolding experiments using the isolated Psm3 CC as a model system. We find a comparatively unstable protein with three unzipping intermediates, which we could directly assign to CC features by crosslinking experiments and state-of-the-art prediction software. Particularly, the CC elbow is shown to be a flexible, potentially non-structured feature, which divides the CC into sections, induces a pairing shift from one CC strand to the other and could facilitate large-scale conformational changes, most likely via thermal fluctuations of the flanking CC sections. A replacement of the elbow amino acids hinders folding of the consecutive CC region and frequently leads to non-native misalignments, revealing the elbow as a guide for proper folding. Additional in vivo manipulation of the elbow flexibility resulted in impaired cohesin complexes, which directly link the sensitive CC architecture to the biological function of cohesin.

Dilution of the cell cycle inhibitor Whi5 controls budding-yeast cell size.

Cell size fundamentally affects all biosynthetic processes by determining the scale of organelles and influencing surface transport. Although extensive studies have identified many mutations affecting cell size, the molecular mechanisms underlying size control have remained elusive. In the budding yeast Saccharomyces cerevisiae, size control occurs in G1 phase before Start, the point of irreversible commitment to cell division. It was previously thought that activity of the G1 cyclin Cln3 increased with cell size to trigger Start by initiating the inhibition of the transcriptional inhibitor Whi5 (refs 6-8). Here we show that although Cln3 concentration does modulate the rate at which cells pass Start, its synthesis increases in proportion to cell size so that its total concentration is nearly constant during pre-Start G1. Rather than increasing Cln3 activity, we identify decreasing Whi5 activity--due to the dilution of Whi5 by cell growth--as a molecular mechanism through which cell size controls proliferation. Whi5 is synthesized in S/G2/M phases of the cell cycle in a largely size-independent manner. This results in smaller daughter cells being born with higher Whi5 concentrations that extend their pre-Start G1 phase. Thus, at its most fundamental level, size control in budding yeast results from the differential scaling of Cln3 and Whi5 synthesis rates with cell size. More generally, our work shows that differential size-dependency of protein synthesis can provide an elegant mechanism to coordinate cellular functions with growth.

Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth.

To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell-cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear-encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

An mTOR/RNA pol I axis shapes chromatin architecture in response to fasting.

Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces a reversible and large-scale spatial reorganization of chromatin in C. elegans . This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, a major regulator of ribosome biogenesis. Remarkably, loss of transcription by RNA Pol I, but not RNA Pol II nor Pol III, induces a similar 3D genome reorganization in fed animals, and prevents the restoration of the fed-state architecture upon restoring nutrients to fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

Live cell microscopy: From image to insight.

Live-cell microscopy is a powerful tool that can reveal cellular behavior as well as the underlying molecular processes. A key advantage of microscopy is that by visualizing biological processes, it can provide direct insights. Nevertheless, live-cell imaging can be technically challenging and prone to artifacts. For a successful experiment, many careful decisions are required at all steps from hardware selection to downstream image analysis. Facing these questions can be particularly intimidating due to the requirement for expertise in multiple disciplines, ranging from optics, biophysics, and programming to cell biology. In this review, we aim to summarize the key points that need to be considered when setting up and analyzing a live-cell imaging experiment. While we put a particular focus on yeast, many of the concepts discussed are applicable also to other organisms. In addition, we discuss reporting and data sharing strategies that we think are critical to improve reproducibility in the field.

Quantitative RNA imaging in single live cells reveals age-dependent asymmetric inheritance.

Asymmetric inheritance of cellular content through cell division plays an important role in cell viability and fitness. The dynamics of RNA segregation are so far largely unaddressed. This is partly due to a lack of approaches to follow RNAs over multiple cellular divisions. Here, we establish an approach to quantify RNA dynamics in single cells across several generations in a microfluidics device by tagging RNAs with the diSpinach aptamer. Using S. cerevisiae as a model, we quantitatively characterize intracellular RNA transport from mothers into their buds. Our results suggest that, at cytokinesis, ENO2 diSpinach RNA is preferentially distributed to daughters. This asymmetric RNA segregation depends on the lifespan regulator Sir2 and decreases with increasing replicative age of mothers but does not result from increasing cell size during aging. Overall, our approach opens more opportunities to study RNA dynamics and inheritance in live budding yeast at the single-cell level.

Fragmentation is crucial for the steady-state dynamics of actin filaments.

Despite the recognition that actin filaments are important for numerous cellular processes, and decades of investigation, the dynamics of in vitro actin filaments are still not completely understood. Here, we follow the time evolution of the length distribution of labeled actin reporter filaments in an unlabeled F-actin solution via fluorescence microscopy. Whereas treadmilling and diffusive length fluctuations cannot account for the observed dynamics, our results suggest that at low salt conditions, spontaneous fragmentation is crucial.

Slow down of actin depolymerization by cross-linking molecules.

The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.

Transcription coordinates histone amounts and genome content.

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates.

Structural and viscoelastic properties of actin networks formed by espin or pathologically relevant espin mutants.

The structural organization of the cytoskeleton determines its viscoelastic response which is crucial for the correct functionality of living cells. Both the mechanical response and microstructure of the cytoskeleton are regulated on a microscopic level by the local activation of different actin binding and/or bundling proteins (ABPs). Misregulations in the expression of these ABPs or mutations in their sequence can entail severe cellular dysfunctions and diseases. Here, we study the structural and viscoelastic properties of reconstituted actin networks cross-linked by the ABP espin and compare the obtained network properties to those of other bundled actin networks. Moreover, we quantify the impact of pathologically relevant espin mutations on the viscoelastic properties of these cytoskeletal networks.

The phenomenology of cell size control.

Cells control their size through an intricate balance of cell growth, cell division, and cell death. Extensive work on unicellular model organisms revealed that cell-size-dependent cell cycle progression accounts for major aspects of cell size regulation and provided insights into the underlying molecular mechanisms. Nevertheless, elaborate live-cell imaging approaches still reveal new phenomenological observations that challenge our simplified models of size regulation and raise the question of what determines optimal cell size. Here, I aim to give a conceptual overview of the many processes contributing to cell size regulation and summarize recent developments in the field.

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

The Biosynthetic Basis of Cell Size Control.

Cell size is an important physiological trait that sets the scale of all biosynthetic processes. Although physiological studies have revealed that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained unclear. Here we review recent progress in identifying the molecular mechanisms of cell size control. We focus on budding yeast, where cell growth dilutes a cell cycle inhibitor to couple growth and division. We discuss a new model for size control based on the titration of activator and inhibitor molecules whose synthesis rates are differentially dependent on cell size.