Tuberin levels during cellular differentiation in brain development.

Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex (TSC), and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, permits the formation of tumors that can lead to developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that total Tuberin levels are tightly regulated through developmental stages in the embryonic brain. At a cell biology level, we show that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. This functional data supports the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies.

Dissecting the roles of the Tuberin protein in the subcellular localization of the G2/M Cyclin, Cyclin B1.

Tuberin is a major component of the protein regulatory complex known as the Tuberous Sclerosis Complex and plays a crucial role in cell cycle progression and protein synthesis. Mutations in the Tuberin gene, TSC2, lead to the formation of benign tumors in many organ systems and causes the Tuberous Sclerosis Complex disorder. Genotypes ranging from point mutations to large deletions in the TSC2 gene have been clinically characterized with a wide range of phenotypes from skin tumors to large brain tumors. Our lab has previously demonstrated that Tuberin can directly bind and regulate the timing of nuclear transport of the G2/M cyclin, Cyclin B1. Herein we study the consequence of one clinically relevant truncation in the Tuberin protein on cell cycle function. We demonstrate that exogenous expression of a fragment of the N-term region of Tuberin alters the subcellular localization of Cyclin B1 and increases cell proliferation. This adds to our body of information about the residues within Tuberin responsible for regulating the cytoplasmic retention of Cyclin B1 and supports the phenotypic data seen in the clinic with Tuberous Sclerosis Complex patients harbouring similar large deletions in Tuberin.

The Tuberin and Cyclin B1 complex functions as a novel G2/M sensor of serum conditions and Akt signaling.

A great deal of ground breaking work has determined that the Tuberin and Hamartin Complex function as a negative regulator of protein synthesis and cell cycle progression through G1/S. This is largely attributed to the GTPase activity of Tuberin that indirectly inhibits the mammalian target of rapamycin (mTOR). During times of ample nutrition Tuberin is inhibited by growth factor signaling, including direct phosphorylation by Akt/PKB, allowing for activation of mTOR and subsequent protein synthesis. It is well rationalized that maintaining homeostasis requires communication between cell growth (mTOR signaling) and cell division (cell cycle regulation), however how this occurs mechanistically has not been resolved. This work demonstrates that in the presence of high serum, and/or Akt signaling, direct binding between Tuberin and the G2/M cyclin, Cyclin B1, is stabilized and the rate of mitotic entry is decreased. Importantly, we show that this results in an increase in cell size. We propose that this represents a novel cell cycle checkpoint linking mitotic onset with the nutritional status of the cell to control cell growth.

Derivation of a novel G2 reporter system.

Progression through G2 phase of the cell cycle is a technically difficult area of cell biology to study due to the lack of physical markers specific to this phase. The FUCCI system uses the biology of the cell cycle to drive fluorescence in select phases of the cell cycle. Similarly, a commercially available system has used a fluorescent analog of the Cyclin B1 protein to visualize cells from late S phase to the metaphase-anaphase transition. We have modified these systems to use the promoter and destruction box elements of Cyclin B1 to drive a cyan fluorescent protein. We demonstrate here that this is a useful tool for measuring the length of G2 phase without perturbing any aspect of cell cycle progression.

The cyclin-like protein Spy1 regulates growth and division characteristics of the CD133+ population in human glioma.

The heterogeneity of brain cancers, as most solid tumors, complicates diagnosis and treatment. Identifying and targeting populations of cells driving tumorigenesis is a top priority for the cancer biology field. This is not a trivial task; considerable variance exists in the driving mutations, identifying markers, and evolutionary pressures influencing initiating cells in different individual tumors. Despite this, the ability to self-renew and differentiate must be conserved to reseed a heterogeneous tumor mass. Focusing on one example of a tumor-initiating cell population, we demonstrate that the atypical cyclin-like protein Spy1 plays a role in balancing the division properties of glioma cells with stemness properties. This mechanistic insight may provide new opportunities for therapeutic intervention of brain cancer.

The tumor suppressor tuberin regulates mitotic onset through the cellular localization of cyclin B1.

Tuberous sclerosis is a multi-organ disorder characterized by the formation of benign tumors, called hamartomas, which affects more than 1 million people worldwide. The syndrome is initiated by a mutation in one of two tumor suppressor genes, TSC1 or TSC2, that encode for the proteins hamartin and tuberin, respectively. Herein, we demonstrate that tuberin binds and regulates the G 2/M cyclin, cyclin B1. We have determined that this binding region encompasses a mutational hotspot within tuberin that is implicated in some of the most severe cases of TS. Mimicking a mutation found in a subset of patients with tuberous sclerosis, we found a significant reduction in the binding between tuberin and cyclin B1. Functionally, our data supports that tuberin plays a role in regulating the cellular localization of cyclin B1. These results demonstrate a novel and clinically relevant mechanism, where tuberin functions in mitotic onset.

DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase.

DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s(-1); (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.

A method to select for mutator DNA polymerase deltas in Saccharomyces cerevisiae.

Proofreading DNA polymerases share common short peptide motifs that bind Mg(2+) in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase delta mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase delta mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.

Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators.

Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase. The rTnC/CaM chimera, in which the carboxyl-terminal domain of TnC is replaced by that of CaM, has the same ability as CaM to bind and transmit the signal to Ca2+ sites on the enzyme. There is no further functional gain when the amino-terminal domain is modified to make the rTnC/CaM chimera more CaM-like. To identify which regions of the carboxyl-terminal domain of CaM are responsible for these effects, we constructed the chimeras rTnC/3CaM and rTnC/4CaM, where only one-half of the C-terminal domain of CaM (residues 85-112 or residues 113-148) replaces the corresponding region in rTnC. Neither rTnC/3CaM nor rTnC/4CaM can mimic CaM in its affinity for the enzyme. Nevertheless, with respect to the signal transduction process, rTnC/4CaM, but not rTnC/3CaM, shows the same behaviour as CaM. We conclude that the whole C-terminal domain is required for binding to the enzyme while Ca2+-binding site 4 of CaM bears all the requirements to increase Ca2+ binding at PMCA sites. Such mechanism of binding and activation is distinct from that proposed for most other CaM targets. Furthermore, we suggest that Ala128 and Met124 from CaM site 4 may play a crucial role in discriminating CaM from TnC.

Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases.

The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.

Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase.

The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.