A longstanding question in biology is whether there is an intrinsic mechanism for coordinating growth and cell cycle in metazoan cells. We have examined cell size distributions in populations of lymphoblasts and applied a novel mathematical analysis to calculate accurately how growth rates vary with both cell size and the cell cycle. Our results show that growth rate is size-dependent throughout the cell cycle. After initial growth suppression there is a rapid increase in growth rate in G1, followed by a constant exponential growth phase. The probability of cell division varies independently with cell size and cell age. We conclude that proliferating mammalian cells have an intrinsic mechanism that maintains cell size.

Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably – apoptosis. The cyanine dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.

We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the ‘instantaneous’ growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.

We introduce a microfluidic system for simultaneously measuring single cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 hours of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1/S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis.

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.uring protein ubiquitination, a lysine residue on a target protein is conjugated to ubiquitin through an enzymatic cascade involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligases (1). Chain formation can proceed in the same manner by repetitively adding ubiquitins to one or more of seven lysines on conjugated ubiquitin, potentially generating ubiquitin chains of several different topologies (2, 3). The linkage, topology, and length of the ubiquitin chains are thought to determine the fate of the tagged protein: K48-linked ubiquitin for degradation and K63 for signaling DNA damage and inflammation (4).We have studied the linkage and topology of ubiquitin chains generated by the APC, a multisubunit E3 enzyme that controls cell-cycle progression (5). Recently, two interesting observations have been made about chain topology: First, different E2s might be required in concert to form long ubiquitin conjugates on APC substrates (6). Second, the density of ubiquitin, rather than the length of the chains, may qualify substrates for degradation (7).Like most E3 ligases, APC modifies multiple lysine residues on each substrate, generating a diverse mixture of products. Poor resolution obtained in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) hinders analysis. Even when distinct bands are observed, they may not correspond to single products. A band of a discrete molecular weight may reflect ubiquitination on multiple lysine residues or, alternatively, the formation of ubiquitin chains on single-lysine sites.Dissecting the topology of the ubiquitin chains requires a careful simplification of the ubiquitination reaction under meaningful conditions. To achieve that, following earlier work of Petroski and Deshaies (8), we have modified the well-characterized APC substrate, securin, and generated a model substrate that has only a single-lysine residue available for ubiquitination, thereby limiting chain formation to a single chain. By using this model substrate, we discovered that the ubiquitin conjugates formed on APC substrates are preferentially linked through ubiquitin lysine 11. Similar results have r...

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work.

The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins-Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and sizedependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.cell division asymmetry | cell growth | cell dry mass | interferometry | synthetic phase microscopy

Using multiplexed quantitative proteomics, we analyzed cell cycledependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/ Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C CDH1 -dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.