Biology subjects

Manalis, S. R.

Publications and source records attributed to Manalis, S. R..

22 records · Page 2Linked to original sources

bioRxiv2020

HSP90 facilitates oncogenic alterations of metabolism in B-cell lymphomas

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here, we described a novel role of HSP90 in the cytosolic compartmentalization of metabolic pathways in proliferating cancer cells. We found that HSP90 assists in the organization of metabolic enzymes into non-membrane-bound functional compartments termed metabosomes. Under experimental conditions that conserved the cellular proteostasis, we demonstrated that the compartmentalizing activity of HSP90 is critical to sustain the coordinated synthesis of multiple metabolites required for energy production, maintenance of the cellular biomass and secretion of immunometabolites. Conversely, inhibition of the nucleating capacity of HSP90 modified the topology of cytosolic metabosomes before protein degradation was apparent decreasing the efficiency of MYC-driven metabolic pathways. Inhibition of HSP90 decreases cancer metabolism in B-cell lymphoma cells and patients providing a novel mechanism of activity for this class of drugs.

cancer biology↗

bioRxiv2019

Mass measurements of polyploid lymphocytes reveal that growth is not size limited but depends strongly on cell cycle

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e. growth efficiency) than intermediate sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size and cell cycle dependent growth is challenging. To decouple and quantify cell size and cell cycle dependent growth effects we monitor growth efficiency of freely proliferating and cycling polyploid mouse lymphocytes with high resolution. To achieve this, we develop large-channel suspended microchannel resonators that allow us to monitor mass of single cells ranging from 40 pg (small diploid lymphocyte) to over 4000 pg, with a resolution ranging from ~1% to ~0.05%. We find that mass increases exponentially with respect to time in early cell cycle but transitions to linear dependence during late S and G2 stages. This growth behavior repeats with every endomitotic cycle as cells grow in to polyploidy. Overall, growth efficiency changes 29% due to cell cycle. In contrast, growth efficiency did not change due to cell size over a 100-fold increase in cell mass during polyploidization. Consistently, growth efficiency remained constant when cell cycle was arrested in G2. Thus, cell cycle is a primary determinant of growth efficiency and increasing cell size does not impose transport limitations that decrease growth efficiency in cultured mammalian cells. Significance statementCell size is believed to influence cell behavior through limited transport efficiency in larger cells, which could decrease the growth rate of large cells. However, this has not been experimentally investigated due to a lack of non-invasive, high-precision growth quantification methods suitable for measuring large cells. Here, we have engineered large versions of microfluidic mass sensors called suspended microchannel resonators in order to study the growth of single mammalian cells that range 100-fold in mass. This revealed that the absolute size of a cell does not impose strict transport or other limitations that would inhibit growth. In contrast to cell size, however, cell cycle has a relatively large influence on growth and our measurements allow us to decouple and quantify the growth effects caused by cell cycle and cell size.

cell biology↗

bioRxiv2019

Time-dynamics of mitochondrial membrane potential reveal an inhibition of ATP synthesis in mitosis

The energetic demands of a cell are believed to increase during mitosis 1-7. As cells transit from G2 into mitosis, mitochondrial electron transport chain (ETC) activity increases 4,8,9, and cellular ATP levels progressively decrease until the metaphase-anaphase transition 3,7,10, consistent with elevated consumption. The rates of ATP synthesis during mitosis, however, have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytes and demonstrate that cyclin-dependent kinase 1 (CDK1) activity causes mitochondrial hyperpolarization from G2/M until the metaphase-anaphase transition. By using an electrical circuit model of mitochondria, we quantify the time-dynamics of mitochondrial membrane potential under normal and perturbed conditions to extract mitochondrial ATP synthesis rates in mitosis. We found that mitochondrial ATP synthesis decreases by approximately 50 % during early mitosis, when CDK1 is active, and increases back to G2 levels during cytokinesis. Consistently, acute inhibition of mitochondrial ATP synthesis failed to delay cell division. Our results provide a quantitative understanding of mitochondrial bioenergetics in mitosis and challenge the traditional dogma that cell division is a highly energy demanding process.

cell biology↗

bioRxiv2018

Linking single-cell measurements of mass, growth rate, and gene expression

We introduce a microfluidic platform that enables single-cell mass and growth rate measurements upstream of single-cell RNA-sequencing (scRNA-seq) to generate paired single-cell biophysical and transcriptional data sets. Biophysical measurements are collected with a serial suspended microchannel resonator platform (sSMR) that utilizes automated fluidic state switching to load individual cells at fixed intervals, achieving a throughput of 120 cells per hour. Each single-cell is subsequently captured downstream for linked molecular analysis using an automated collection system. From linked measurements of a murine leukemia (L1210) and pro-B cell line (FL5.12), we identify gene expression signatures that correlate significantly with cell mass and growth rate. In particular, we find that both cell lines display a cell-cycle signature that correlates with cell mass, with early and late cell-cycle signatures significantly enriched amongst genes with negative and positive correlations with mass, respectively. FL5.12 cells also show a significant correlation between single-cell growth efficiency and a G1-S transition signature, providing additional transcriptional evidence for a phenomenon previously observed through biophysical measurements alone. Importantly, the throughput and speed of our platform allows for the characterization of phenotypes in dynamic cellular systems. As a proof-of-principle, we apply our system to characterize activated murine CD8+ T cells and uncover two unique features of CD8+ T cells as they become proliferative in response to activation: i) the level of coordination between cell cycle gene expression and cell mass increases, and ii) translation-related gene expression increases and shows a correlation with single-cell growth efficiency. Overall, our approach provides a new means of characterizing the transcriptional mechanisms of normal and dysfunctional cellular mass and growth rate regulation across a range of biological contexts.

bioengineering↗