The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.cell-cell interaction | intercellular communication | surface acoustic waves | acoustic tweezers | acoustofluidics M ulticellular systems rely on the interaction between cells to coordinate cell signaling and regulate cell functions. Understanding the mechanism and process of cell-cell interaction is critical to many physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes (1-3). Despite significant advances in this field, to further understand how cells interact and communicate with each other, a robust, biocompatible method to precisely control the spatial and temporal association of cells and to create defined cellular assemblies is urgently needed (4). Although several methods have been used to pattern cells, limitations still exist for the demonstrated methods including those that make use of optical, electrical, magnetic, hydrodynamic, and contact printing technologies (5-9). Firstly, most of the methods require modification of the cell's native state. The magnetic assembly method, for example, requires cells to be labeled with magnetic probes. Dielectrophoresis typically requires the use of a special medium (e.g., nonconductive) which may lack essential nutrients or have biophysical properties (such as the osmolality) that may adversely affect cell growth or physiology (6). Optical tweezers provide a label-free and contactless approach, but typically require high laser power to manipulate cells, leading to a high risk of cell damage (5). Secondly, the working principles of the existing technologies mostly preclude the combination of high precision and high throughput into a single ...
Purine biosynthetic enzymes organize into dynamic cellular bodies called purinosomes. Little is known about the spatiotemporal control of these structures. Using super-resolution microscopy, we demonstrated that purinosomes colocalized with mitochondria, and these results were supported by isolation of purinosome enzymes with mitochondria. Moreover, the number of purinosome containing cells responded to dysregulation of mitochondrial function and metabolism. To explore the role of intracellular signaling, we performed a kinome screen using a label-free assay and identified that mTOR influenced purinosome assembly. mTOR inhibition disrupted purinosome-mitochondria colocalization and suppressed purinosome formation stimulated by mitochondria dysregulation. Collectively, our data suggests an mTOR-mediated link between purinosomes and mitochondria and suggests a general means by which mTOR regulates nucleotide metabolism by spatiotemporal control over protein association.
Background: Metabolic enzymes have been hypothesized to assemble into complex to respond to cellular metabolism changes. Results: De novo purine biosynthesis increases in purinosome-containing cells. Conclusion: Purine metabolism is adjusted by purinosome assembly. Significance: This study indicates that purinosome is a functional multienzyme complex.
The de novo purine biosynthetic pathway relies on six enzymes to catalyze the conversion of phosphoribosylpyrophosphate to inosine 5′-monophosphate. Under purine-depleted conditions, these enzymes form a multienzyme complex known as the purinosome. Previous studies have revealed the spatial organization and importance of the purinosome within mammalian cancer cells. In this study, time-lapse fluorescence microscopy was used to investigate the cell cycle dependency on purinosome formation in two cell models. Results in HeLa cells under purine-depleted conditions demonstrated a significantly higher number of cells with purinosomes in the G 1 phase, which was further confirmed by cell synchronization. HGPRT-deficient fibroblast cells also exhibited the greatest purinosome formation in the G 1 phase; however, elevated levels of purinosomes were also observed in the S and G 2 /M phases. The observed variation in cell cycle-dependent purinosome formation between the two cell models tested can be attributed to differences in purine biosynthetic mechanisms. Our results demonstrate that purinosome formation is closely related to the cell cycle.
a b s t r a c tSGs are mRNA containing cytoplasmic structures that are assembled in response to stress. Tudor-SN protein is a ubiquitously expressed protein. Here, Tudor-SN protein was found to physiologically interact with G3BP, which is the marker and effector of SG. The kinetics of the assembly of SGs in the living cells demonstrated that Tudor-SN co-localizes with G3BP and is recruited to the same SGs in response to different stress stimuli. Knockdown of endogenous Tudor-SN did not inhibit the formation of SGs, but retarded the aggregation of small SGs into large SGs. Thus Tudor-SN may not be an initiator as essential as G3BP for the formation of SGs, but affects the aggregation of SGs. These findings identify Tudor-SN as a novel component of SGs. Structured summary:MINT-7968768, MINT-7968779: Tudor-SN (uniprotkb:Q7KZF4) physically interacts (MI:0915) with G3BP
The de novo biosynthesis of purines is carried out by a highly conserved metabolic pathway that includes several validated targets for anticancer, immunosuppressant, and anti-inflammatory chemotherapeutics. The six enzymes in humans that catalyze the 10 chemical steps from phosphoribosylpyrophosphate to inosine monophosphate were recently shown to associate into a dynamic multiprotein complex called the purinosome. Here, we demonstrate that heat shock protein 90 (Hsp90), heat shock protein 70 (Hsp70), and several cochaperones functionally colocalize with this protein complex. Knockdown of expression levels of the identified cochaperones leads to disruption of purinosomes. In addition, small molecule inhibitors of Hsp90 and Hsp70 reversibly disrupt purinosomes and are shown to have a synergistic effect with methotrexate, an anticancer agent that targets purine biosynthesis. These data implicate the Hsp90/Hsp70 chaperone machinery in the assembly of the purinosome and provide a strategy for the development of improved anticancer therapies that disrupt purine biosynthesis.purine metabolism | J-domain protein | Bcl-2-associated anthogene domain protein
Purine nucleotides are ubiquitous molecules that play vital roles in all kingdoms of life, not only as components of nucleic acids, but also participating in signaling and energy storage. Cellular pools of purines are maintained by the tight control of several complementary and sometimes competing processes including de novo biosynthesis, salvage and catabolism of nucleotides. While great strides have been made over the past sixty years in understanding the biosynthesis of purines, we are experiencing a renaissance in this field. In this feature article we discuss the most recent discoveries relating to purine biosynthesis, with particular emphasis upon the dynamic multi-protein complex called the purinosome. In particular we highlight advances made towards understanding the assembly, control and function of this protein complex and the attempts made to exploit this knowledge for drug discovery.
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