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Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites., Sir Henry Wellcome Fellowship (103972/Z/14/Z), European Research Council (research grant ERC- 2012-StG 309255-EndoTox), National Institutes of Health (U.S.) (NIH Exploratory R21 grant 1R21AI123746), United States. Army Research Office. Institute for Collaborative Biotechnologies (grant W911NF-09-0001)

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology., National Institutes of Health (U.S.) (Contract R21AI110787), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Science Foundation (U.S.). Graduate Research Fellowship, National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award F32CA1800586), Kinship Foundation. Searle Scholars Program, Beckman Young Investigator Program, National Institutes of Health (U.S.) (New Innovator Award DP2 OD020839)

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated., National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Institutes of Health (U.S.) (Contract R01GM085457), National Cancer Institute (U.S.) (Fellowship F31CA167872), National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Program 5T32GM008334)

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines., Janssen Pharmaceutical Ltd., National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), MIT-Harvard Center of Cancer Nanotechnology Excellence (Grant 26697290-47281-A), Stand Up To Cancer, National Institutes of Health (U.S.). P41 Biotechnology Resource Center, National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051)

Therapeutics that block kinases, transcriptional modifiers, immune checkpoints and other biological vulnerabilities are transforming cancer treatment. As a result, many patients achieve dramatic responses, including complete radiographical or pathological remission, yet retain minimal residual disease (MRD), which results in relapse. New functional approaches can characterize clonal heterogeneity and predict therapeutic sensitivity of MRD at a single-cell level. Preliminary evidence suggests that iterative detection, profiling and targeting of MRD would meaningfully improve outcomes and may even lead to cure., Koch Institute-Dana-Farber/Harvard Cancer Center Bridge Project, National Cancer Institute (U.S.) (R33 CA191143), National Cancer Institute (U.S.). Cancer Systems Biology Consortium (U54 CA217377)

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes., United States. Army Research Office (Grant W911NF-09-D-0001), National Science Foundation (U.S.) (Grant 1129359), National Cancer Institute (U.S.) (Grant U54CA143874), National Cancer Institute (U.S.) (Grant P30-CA14051), National Cancer Institute (U.S.) (Grant R33-CA191143), National Institutes of Health (U.S.) (Grant T32-GM008334), National Institute of General Medical Sciences (U.S.) (Grant T32-GM008334)

Simultaneously measuring multiple eigenmode frequencies of nanomechanical resonators can determine the position and mass of surface-adsorbed proteins, and could ultimately reveal the mass tomography of nanoscale analytes. However, existing measurement techniques are slow (<1 Hz bandwidth), limiting throughput and preventing use with resonators generating fast transient signals. Here we develop a general platform for independently and simultaneously oscillating multiple modes of mechanical resonators, enabling frequency measurements that can precisely track fast transient signals within a user-defined bandwidth that exceeds 500 Hz. We use this enhanced bandwidth to resolve signals from multiple nanoparticles flowing simultaneously through a suspended nanochannel resonator and show that four resonant modes are sufficient for determining their individual position and mass with an accuracy near 150 nm and 40 attograms throughout their 150-ms transit. We envision that our method can be readily extended to other systems to increase bandwidth, number of modes, or number of resonators., United States. Army Research Office (Grant W911NF-09-0001), Center for Integration of Medicine and Innovative Technology (Contract 09-440), National Science Foundation (U.S.) (Grant 1129359)

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 h 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., National Cancer Institute (U.S.) (Physical Sciences Oncology Center, U54CA143874), National Cancer Institute (U.S.) (Physical Sciences Oncology Center, R21 CA137695), National Institute of General Medical Sciences (U.S.) ((NIGMS) EUREKA R01GM085457), Kwanjeong Educational Foundation (Korea) (Graduate Fellowship), National Science Foundation (U.S.) (Graduate Research Fellowship), European Commission. Community Research and Development Information Service (Marie Curie International Reintegration Grant PIRG-GA-2010-277062), Israeli Centers of Research Excellence (I-CORE) (Program, Center no. 41/11)

Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell’s biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean., National Science Foundation (U.S.) (OCE-1129359), Simons Foundation (337262), United States. Army Research Office (W911NF-09-D-0001)

Multiple myeloma (MM) has benefited from significant advancements in treatment that have improved outcomes and reduced morbidity. However, the disease remains incurable and is characterized by high rates of drug resistance and relapse. Consequently, methods to select the most efficacious therapy are of great interest. Here we utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring their mass accumulation rate (MAR). We show that MAR accurately and rapidly defines therapeutic susceptibility across human multiple myeloma cell lines to a gamut of standard-of-care therapies. Finally, we demonstrate that our MAR assay, without the need for extended culture ex vivo, correctly defines the response of nine patients to standard-of-care drugs according to their clinical diagnoses. This data highlights the MAR assay in both research and clinical applications as a promising tool for predicting therapeutic response using clinical samples.

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., National Institutes of Health (U.S.) (MIT Center for Cell Decision Processes Grant P50GM68762), United States. Army Research Office (Institute for Collaborative Biotechnologies Grant DAAD1903D0004)