Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/CCdh1APC/C^{Cdh1} Complex

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the $APC/C^{Cdh1}$ complex, but not by the $APC/C^{Cdc20}$ complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division

Direct observation of mammalian cell growth and size regulation

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

Optimizing Optical Flow Cytometry for Cell Volume-Based Sorting and Analysis

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

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Identifying attributes of public transport services for urban tourists: A data-mining method

The current work focuses on Quality of Service (QoS) of Public Transport (PT) attributes in urban tourist destinations. In particular, we aim to reveal which attributes are most significant for tourists prior to their arrival at their destination, as reflected in questions posted in TripAdvisor Question and Answer forums, a widely used social media platform. We used a data-mining method to classify questions into categories relevant to QoS, using a sample of 8905 items posted between 2005 and 2018 in TripAdvisor forums for seven urban destinations in the United States and Western Europe. We found four PT-QoS attributes: Pricing and ticketing, Accessibility, Trip duration, and Service availability (hours of operation and frequency). These attributes have similar relative significance for all destinations, origins, seasons, and years we checked. Hence, they can help service operators and policymakers to understand tourists\u27 preferences and to adjust PT services accordingly

Using Standard Optical Flow Cytometry for Synchronizing Proliferating Cells in the G1 Phase

<div><p>Cell cycle research greatly relies on synchronization of proliferating cells. However, effective synchronization of mammalian cells is commonly achieved by long exposure to one or more cell cycle blocking agents. These chemicals are, by definition, hazardous (some more than others), pose uneven cell cycle arrest, thus introducing unwanted variables. The challenge of synchronizing proliferating cells in G1 is even greater; this process typically involves the release of drug-arrested cells into the cycle that follows, a heterogeneous process that can truly limit synchronization. Moreover, drug-based synchronization decouples the cell cycle from cell growth in ways that are understudied and intolerable for those who investigate the relationship between these two processes. In this study we showed that cell size, as approximated by a single light-scatter parameter available in all standard sorters, can be used for synchronizing proliferating mammalian cells in G1 with minimal or no risk to either the cell cycle or cell growth. The power and selectivity of our method are demonstrated for human HEK293 cells that, despite their many advantages, are suboptimal for synchronization, let alone in G1. Our approach is readily available, simple, fast, and inexpensive; it is independent of any drugs or dyes, and nonhazardous. These properties are relevant for the study of the mammalian cell cycle, specifically in the context of G1 and cell growth.</p></div

Size-based sorting of L1210 and A549 cells yields G1 cell populations.

<p>L1210 exhibiting the lowest 2.5% of the FSC-W distribution were gated for sorting (FACSAria III). Similarly, A549 cells exhibiting the lowest 10% of the SSC-A distribution were gated for sorting. Post-sorted and pre-sorted (unsynchronized) L1210 and A549 cells were then fixed and stained with PI for quantification of DNA and cell cycle progression (ModFit LT), which is also shown.</p

A schematic illustrating the continuous growth of a proliferating cell throughout its life cycle.

<p>(A) The overall correlation between geometric size (cell volume) and time from birth enables, in principle, size-based cell cycle synchronization of proliferating cells. (B) Because of the inherent cell-to-cell variability in both, growth rate (cell size over time) and cell cycle progression (also known as the dispersion phenomenon), average cell size, <i>s</i>, best correlates with the cell cycle in small rather than large cells (illustrated by the three red circles [top] and the size distributions). The smallest cells are, on the average, the youngest ones (<i>i.e.,</i> in G1 phase). The various cell cycle phases are depicted by single letter codes: G1, S, G2, and M.</p

Size-based sorting can be utilized for nonperturbative cell cycle synchronization in the G1 phase.

<p>(A) HEK293 cells exhibiting the 8% lowest FSC-W intensity were sorted (FACSAria III) and incubated in fresh, warm media for up to 44 hrs. Cells were harvested at the indicated time points, fixed, and stained with PI for quantifying DNA (Gallios) and cell cycle progression (ModFit LT). DNA quantification of pre-sorted, unsynchronized HEK293 cells (unsync) is also shown. (B) HEK293 cells were synchronized in prometaphase, following the thymidine/nocodazole block. Cells were harvested for PI staining or washed, re-cultured for up to 7 hr, and then harvested. We used the ModFit LT software to assess cell synchronization and cell cycle progression. (C) HEK293 cells were sorted as described in A, and re-cultured immediately in fresh, warm media. Cell volume distribution was measured at 0, 1.5, 3, and 5 hrs post-sorting using the Multisizer IV Coulter counter (see color code on the right). Median values of the four volume distributions are tabled (right).</p

Optimizing size-based sorting of HEK293 cells.

<p>(A) The low and high 10% ends of the depicted light-scatter distributions of proliferating HEK293 cells were gated for sorting by FACSAria III. (B) Volume distributions of the sorted low- and high-end cell populations, as determined by Multisizer IV Coulter counter, are plotted in red and black, respectively. We used the calculated percent overlap and the difference in median values (Δ median) between the measured volume distributions of the ‘small’ and ‘large’ sorted cell populations to evaluate the competency of various light-scatter parameters to approximate cell size in HEK293.</p