Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division.

Cell size is fundamental to cell physiology. For example, cell size determines the spatial scale of organelles and intracellular transport and thereby affects biosynthesis. Although some genes that affect mammalian cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive. We show that cell growth during the G1 phase of the cell division cycle dilutes the cell cycle inhibitor Retinoblastoma protein (Rb) to trigger division in human cells. RB overexpression increased cell size and G1 duration, whereas RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of the G1 phase. Thus, Rb dilution through cell growth in G1 provides one of the long-sought molecular mechanisms that promotes cell size homeostasis.

Constitutive expression of a fluorescent protein reports the size of live human cells.

Cell size is important for cell physiology because it sets the geometric scale of organelles and biosynthesis. A number of methods exist to measure different aspects of cell size, but each has significant drawbacks. Here, we present an alternative method to measure the size of single human cells using a nuclear localized fluorescent protein expressed from a constitutive promoter. We validate this method by comparing it to several established cell size measurement strategies, including flow cytometry optical scatter, total protein dyes, and quantitative phase microscopy. We directly compare our fluorescent protein measurement with the commonly used measurement of nuclear volume and show that our measurements are more robust and less dependent on image segmentation. We apply our method to examine how cell size impacts the cell division cycle and reaffirm that there is a negative correlation between size at cell birth and G1 duration. Importantly, combining our size reporter with fluorescent labeling of a different protein in a different color channel allows measurement of concentration dynamics using simple wide-field fluorescence imaging. Thus, we expect our method will be of use to researchers interested in how dynamically changing protein concentrations control cell fates.

LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

The mRNA processing body (P-body) is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB), the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex) co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

Small, highly active DNAs that hydrolyze DNA.

DNA phosphoester bonds are exceedingly resistant to hydrolysis in the absence of chemical or enzymatic catalysts. This property is particularly important for organisms with large genomes, as resistance to hydrolytic degradation permits the long-term storage of genetic information. Here we report the creation and analysis of two classes of engineered deoxyribozymes that selectively and rapidly hydrolyze DNA. Members of class I deoxyribozymes carry a catalytic core composed of only 15 conserved nucleotides and attain an observed rate constant (k(obs)) of ~1 min(-1) when incubated near neutral pH in the presence of Zn(2+). Natural DNA sequences conforming to the class I consensus sequence and structure were found that undergo hydrolysis under selection conditions (2 mM Zn(2+), pH 7), which demonstrates that the inherent structure of certain DNA regions might promote catalytic reactions, leading to genomic instability.

Insulin analogs for the treatment of diabetes mellitus: therapeutic applications of protein engineering.

The engineering of insulin analogs represents a triumph of structure-based protein design. A framework has been provided by structures of insulin hexamers. Containing a zinc-coordinated trimer of dimers, such structures represent a storage form of the active insulin monomer. Initial studies focused on destabilization of subunit interfaces. Because disassembly facilitates capillary absorption, such targeted destabilization enabled development of rapid-acting insulin analogs. Converse efforts were undertaken to stabilize the insulin hexamer and promote higher-order self-assembly within the subcutaneous depot toward the goal of enhanced basal glycemic control with reduced risk of hypoglycemia. Current products either operate through isoelectric precipitation (insulin glargine, the active component of Lantus(®); Sanofi-Aventis) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir(®); Novo-Nordisk). To further improve pharmacokinetic properties, modified approaches are presently under investigation. Novel strategies have recently been proposed based on subcutaneous supramolecular assembly coupled to (a) large-scale allosteric reorganization of the insulin hexamer (the TR transition), (b) pH-dependent binding of zinc ions to engineered His-X(3)-His sites at hexamer surfaces, or (c) the long-range vision of glucose-responsive polymers for regulated hormone release. Such designs share with wild-type insulin and current insulin products a susceptibility to degradation above room temperature, and so their delivery, storage, and use require the infrastructure of an affluent society. Given the global dimensions of the therapeutic supply chain, we envisage that concurrent engineering of ultra-stable protein analog formulations would benefit underprivileged patients in the developing world.