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Experiments in Cell Biology

What follows is a series of potential experimental ideas and angles that may be of interest to a cell biologist. The range of topics is fairly broad and covers a number of areas ranging from protein degradation and trafficking all the way to stem cell identification and functionality. While some of these experiments may have already been conducted, others are currently poorly researched or simply not possible, making them valuable targets for future study.

Autophagy

One possible autophagic mechanism involved in the elimination of this oxidized target protein is the selective process of chaperone-mediated autophagy (CMA). During CMA, cytosolic chaperones including HSC70 bind sequences on substrate proteins and associate with the cytosolic face of the lysosomal membrane protein LAMP2A. Substrate accumulation leads to LAMP2A multimerization, forming a translocon through which the substrate protein passes, allowing it to be degraded in the lysosome. An alternative degradative system is the non-specific macroautophagy pathway. This process is complex, but briefly the proteins ULK1/2 nucleate a complex containing many other proteins which recruit a segment of membrane that is elongated by Atg-family proteins, engulfing a portion of cytoplasm. This newly formed autophagosome ultimately fuses with a lysosome, allowing for the degradation of intravesicular proteins.

Because CMA is a selective process, an initial line of investigation should include an examination of the sequence of the oxidized protein for a KFERQ-homology sequence (either by direct sequencing or by probing oxidized forms of the protein with polyclonal anti-KFERQ antibodies). If such a sequence is present on this protein it is a good candidate for CMA. To confirm this result, cells lacking LAMP2A should be monitored for accumulation of the oxidized protein as assessed by Western blotting, protein-specific ELISA, or by quantification of cytosolic and lysosomal protein levels. Because LAMP2A is essential for CMA but not for macroautophagy, accumulation of the oxidized protein in LAMP2A-/- cells would suggest that CMA is the primary mechanism of autophagic degradation. Similarly, to test whether macroautophagy is responsible for protein degradation, protein accumulation can be monitored in cells depleted of ULK1/2 using siRNA. Alternatively cells can be transfected with influenza A matrix protein 2 which blocks autophagosome fusion with lysosomes, providing an alternate means of monitoring oxidized protein accumulation in the absence of macroautophagy [1].

Heavy oxidation of proteins can lead to their cytosolic accumulation, creating large aggregates which may be resistant to proteasomal degradation. Conversely, oxidation can make proteins better able to translocate into the lysosome, possibly due to partial unfolding that allows them to more easily pass through the LAMP2A translocon [2]. As such, cells may preferentially degrade this oxidized protein via CMA due to the reduced energy cost associated with this degradation process during times of oxidative stress in the cell when energy likely needs to be diverted to protective mechanisms.

Multi Component Signaling

One clear advantage to a complex multi-component signaling pathway is the fact that a complex pathway has the potential to better amplify a small, transient extracellular sample in order to produce a robust cellular response. If each level of a theoretical hierarchical signaling cascade were to activate twice as many signaling molecules than the previous level, then a single receptor in a three component pathway would ultimately activate only 8 terminal signals. In a ten component cascade, however, a single receptor would activate 1024. While these numbers are not realistic and true signaling pathways are more complex, it remains true that a more robust response can potentially be generated by complex signaling cascades than by simple ones in response to comparable amounts of signal.

Another advantage of a complex system is that an increase in the number of components offers several means by which the response can be regulated. If a signal transduction pathway were composed of three components acting in a stepwise manner then only these three components could be targeted by regulatory mechanisms, likely assuring that the response only be “on” or “off”. In contrast, a complex system with multiple parallel mechanisms and myriad components could be regulated at many points, fine tuning signaling events so as to better direct the intended response based on specific intracellular and extracellular stimuli.

While an abundance of signaling components may allow for fine regulation of the signaling cascade, one disadvantage to a complex pathway is the fact that the larger number of activated signal molecules offer more instances for misregulation and disruption of aspects of this pathway. For example, if one pathway were to use four components and another were to use twenty then in cells exposed to mutagenic stress the complex pathway would be five times more likely to be disrupted by mutagenesis than the simple pathway (assuming an equal chance of disruptive mutations for all genes).

A final significant disadvantage of a complex signaling pathway is that signal termination has the potential to be both more time consuming and less energy efficient than for a simple pathway. If signal termination were to require a blockade of a protein which is activated early in the pathway then it would take time for this blockade to disseminate through the signaling cascade, terminating the signal. In contrast, because a simple pathway has few signaling intermediates this signal termination can occur more rapidly. Likewise, if a pathway terminates signal by deactivating all signaling intermediates then this process will be far more energetically costly in a complex pathway (as will the costs of activating these signals initially).

Formins

As suggested, a valuable first line of investigation would be to establish whether these formins function in a cell- or tissue-specific manner. In order to experimentally determine expression of the 15 different formin genes in a wide range of tissues, tissue samples should be harvested from mice and homogenized, and specific cell types of interest should be isolated using magnetic-activated cell sorting. These cells should then be lysed and RNA samples from these cells should be assessed by qRT-PCR in order to determine the relative expression levels of the 15 formin genes in each cell type. Cells which demonstrate significant increases in a particular formin can then have this result validated by means of immunofluorescent detection of the particular formins in association with actin filaments in order to confirm that the alterations in RNA levels correspond to true increases in protein expression. If possible, it would additionally be valuable to repeat these experiments with cells exposed to stress conditions (ie. oxidative stress) or particular extracellular matrix proteins in order to assess whether particular formins are involved in cellular responses to specific stimuli.

Having identified particular cell types and/or conditions in which specific formins are utilized, the next set of experiments should seek to correlate formin localization with activity. Fluorescent co-localization of specific formins and actin filaments in cells expressing them in abundance would allow for one to predict if specific formins are involved in particular actin structures (ie. stress fibers). After associating formins with particular actin structures, one should create tissue-specific knockdowns of formins in the cell types in which they are expressed. The actin networks can be visualized in these cells to looks for alterations corresponding to a lack of particular formins, with particular attention being paid to the structures identified as targets of these formins via co-localization studies. Formins are known to play roles in both actin filament nucleation and elongation; as such, it will additionally be valuable to identify whether a lack of a particular formin can alter actin dynamics both in vivo and in vitro [1]. An in vitro analysis should be conducted by combining defined actin binding proteins and specific formins to a mixture of actin subunits and measuring various parameters including average rates of actin polymerization, branching, and subunit turnover, as well as average filament length. As formins likely play a more dynamic role in vivo, these dynamics should be assessed in live cells expressing particular formins relative to formin-knockdown cells in order to observe formin-dependent changes in actin structures.

DNA Damage Checkpoints

The DNA damage checkpoint prior to G1-S phase transition uses reversible mechanisms to limit damaged cell growth. DNA damage activates the ATM/ATR kinases, which activate the Chk1/Chk2 kinases, leading to p53 stabilization and production of p21 which inhibits the G1/S-Cdk and S-Cdk complexes thus preventing cell cycle progression. If DNA damage is repaired then this arrest is revered and the cell cycle can proceed. This mechanism presumably evolved as a means of limiting growth of mutated cells. If DNA damage triggered an irreversible block in the cell cycle then a majority of cells would die under unfavorable conditions. By instead giving the cell a chance to repair DNA damage, this reversible checkpoint enables a larger proportion of cells to survive in an evolutionarily favorable manner.

The G1-S transition is an irreversible step after which cells are committed to cell cycle progression. If conditions are favorable, the ubiquitin ligase SCFSkp2 targets cyclin inhibitors for irreversible proteasomal degradation leading to Cdk4/cyclin D accumulation. This drives the activation of E2F, which the promotes its own transcription creating a positive feedback loop which further commits the cell to G1-S transition and leads to Cdk2/cyclin E accumulation. From an evolutionary perspective the irreversible nature of this checkpoint is essential; if cells were to replicate their chromosomes in S phase and then regress to the G1 phase then these cells would become polyploid. Daughter cells would then receive an altered number of chromosomes, leading to misregulation of various signaling processes and possibly resulting in cell death.

To assess whether DNA damage mechanisms play a role in cell cycle arrest in E. histolytica, one should first establish whether DNA damage can trigger a state of arrest in these cells. To determine this, cells should be exposed to a range of doses of X-ray radiation, pulsed with BrdU and propidium iodide and then fixed after a range of times, treated with anti-BrdU antibody, and assessed by flow cytometry to determine whether cells have entered S phase (as evidenced by BrdU incorporation [1]. To identify genes altered during this arrested growth phase, DNA microarrays should next be utilized to identify differentially transcribed genes in growth-arrested cells relative to normally growing cells. As a final experiment, bacteria should be transfected with defective forms of the proteins identified in this screen, X-ray irradiated, and assessed for DNA synthesis/cell cycle progression as above. If knockdown of specific proteins can allow cells to progress through the cell cycle despite accumulated DNA damage then this would suggest that these cells utilize a DNA damage checkpoint analogous to the p53 checkpoint in eukaryotic cells.

The Extracellular Matrix

This altered extracellular matrix (ECM) protein is likely functionally similar to fibronectin, explaining its varied role in cancer progression. Early/benign tumors likely decrease secretion of this protein (or expression of its respective integrin) as a means of becoming anchorage independent, allowing the cell to replicate without the limitations normally imposed by ECM proteins released by surrounding cells. Indeed, there is previous evidence suggesting that upregulation of fibronectin by tumor cells can correspond with an onset of malignancy due to cells receiving signals through the α5β1 which can promote angiogenesis [1]. Just as an inability to adhere to this particular ECM protein may enable benign cancer cells to grow, an inability of normal stromal cells to bind to this same protein may lead to a misregulation of organ size. Integrin signaling in response to the ECM regulates cell growth and cytoskeletal organization, and cells which cannot receive the necessary signals to stop growth are thus likely to keep dividing and produce aberrantly large organs, much as benign tumors continue to divide when surrounding cells do not. Hence this mutation alone would be insufficient to promote tumor progression; it would allow cells to proliferate more than they normally would, but it would not become invasive without additional mutations such as p53 misregulation and secretion of this fibronectin-like protein.

In order to demonstrate that this ECM protein can signal through integrins to promote tumor metastasis in pre-malignant cells, one should first chemically induce tumorigenesis in mice lacking the protein of interest. Pre-malignant tumors should be isolated from these mice and transplanted into mice which have a tissue-specific over-expression of this particular ECM protein (with the tumor being transplanted into the protein over-expressing organ). Rates of metastasis and indicators of tumor progression should be monitored in these mice relative to the knockout mice to indicate as to whether expression of this protein by surrounding cells can drive metastasis of a pre-malignant tumor that doesn't express this protein itself. Additionally, to demonstrate that integrin signaling is what transduces the signal generated by this ECM protein into tumor cells, tumors should be isolated as above and treated with siRNA for specific integrins and downstream signaling components before being transplanted into the abovementioned mice. If these tumors are able to metastasize better in the presence of an abundance of this ECM protein but are only able to do so in an integrin-dependent manner then this would support this hypothesis regarding the function of this ECM protein.

Adult Stem Cells

In order to demonstrate that the heart contains adult stem cells (ASCs), an important first experiment would be to collect the heart of an adult mouse and break apart cell-cell adhesions using gentle detergents. These cells should then be plated and pulsed with BrdU; because terminally differentiated cardiomyocytes do not progress through the cell cycle whereas stem cells are capable of undergoing self-renewal, it will thus be possible to identify ASCs and other proliferating cells. Growing candidate ASCs can then be separated from remaining myocytes by differential centrifugation, and they can further be sorted by FACS for expression of characteristic stem cell markers c-kit+ Lin_ [1]. Sorted ASCs can then be plated as single cells in order to grow clonal populations for further analysis. True ASCs should be able to continuously divide for a time, and populations can be monitored for differentiation into cardiomyocytes in vitro as such differentiation would further confirm that these act as cardiac stem cells.

While isolating cells from adult cardiac muscle that express stem cell markers and are able to continuously divide ex vivo suggests the presence of heart ASCs, a functional analysis of the ability of these cells to differentiate is essential. In order to demonstrate that these putative stem cells can differentiate into cardiomyocytes in vivo, candidate heart ASCs should be collected from C57/B6 mice constitutively expressing GFP or another fluorescent marker. These cells should then be injected intravenously or into the heart of GFP _ C57/B6 mice which have suffered from a myocardial infarction injury. After 2 weeks mice should be sacrificed and cardiac sections should be collected for histological analysis. The remaining cardiac muscle should be lysed as above and cells should be fixed and analyzed by flow cytometry for GFP+ cardiomyocytes using cardiomyocyte nuclear markers Nkx2.5 and GATA4 [2]. Histological sections should be analyzed for these same cell markers and for phyiscal characteristics consistent with cardiomyocyte morphology and tissue regeneration following injury. The presence of GFP+ cardiomyocytes in a GFP_ mouse would confirm the regenerative capacity of these cardiac ASCs, demonstrating that they while they can be maintained as an undifferentiated population of stem-like cells they can differentiate into functional terminally differentiated cardiac cells in vivo in order to regenerate the heart following injury.

In the non-centromeric regions of cells overexpressing CENP-A, the protein likely to be displaced by CENP-A is histone H3 as this is the nucleosomal protein that is replaced by CENP-A along the chromatin of centromeric regions.

Centromere Regulation

Because CENP-A is important for directing the formation of centromeres, CENP-A over-expression has the potential to induce chromosomal instability due to neo-centromere formation at multiple sites on a single chromosome. Newly formed centromeres/kinetochores can result in multipolar spindle formation, improper chromosome segregation, aneuploidy, and subsequent cell misregulation and/or death. The formation of kinetochores at these sites is mediated by a number of factors which associate with CENP-A, including the protein CENP-C. CENP-C binds to CENP-A rich regions of DNA and serves as a platform which recruits several different proteins which ultimately lead to kinetochore formation, thus making CENP-C essential to CENP-A induced neo-centromere formation and consequent aneuploidy (2).

Both boundary regions and centromeres are regions in which very little chromatin remodeling should be occurring due to the heterochromatin content of these sites. Both boundary regions and centromeres contain inverted repeat elements which, along with siRNA and heterochromatin, are essential for CENP-A insertion and centromere formation. The mechanism underlying this process has not been fully defined, but the heterochromatin spreading protein HP1 is speculated to form a complex with small RNAs generated by inverted repeat elements, and the resultant RNA interference is essential for nearby heterochromatin formation and for CENP-A insertion (1). While the HP1 machinery is typically involved in the spread of heterochromatin throughout the genome, siRNA corresponding to gene segments near boundary regions may allow it to thereby participate in the preferential insertion of CENP-A at these sites.

If CENP-A overexpression were repressed back to WT levels, one would expect that over the subsequent generations the cells would begin to lose their mislocalized CENP-A and any de novo kinetochores that formed thereupon. This would be expected because even though CENP-A is epigenetically maintained at centromeres, its insertion in centromeric nucleosomes seems to be favored such that the limited pool of CENP-A would be exhausted following its insertion into proper centromeres, meaning that little if any would remain to propagate the new centromeres which formed as a result of CENP-A overexpression.

TING mRNA Regulation

TING mRNA stability could be regulated by an AU-rich element (ARE) in the 3' UTR. AREs are bound by a variety of binding protein factors such as Hu family proteins which increase mRNA stability or TTP which binds certain ARE motifs and induces deadenylation, resulting in degradation of the poly-A tail and consequent mRNA destabilization. Alternatively, this mRNA could be regulated by an iron response element (IRE) consisting of a particular stem loop structure in the 3' UTR. IRE stem loops are recognized and bound by iron regulatory proteins 1/2 (IRP1/2), stabilizing the mRNA. IRP stability is mediated by the levels of intracellular iron such that these proteins are only active when iron levels are low, allowing for selective stabilization of IRE-containing mRNA in response to iron starvation. A third possibility is that TING mRNA is regulated by an endogenous miRNA or siRNA. These miRNAs associate with Argonaute in the RNA-induced silencing complex (RISC) and bind to mRNA displaying complementary nucleotide sequences. If base pairing is imperfect, the mRNA will be translationally repressed and deadenylated, leading to degradation, whereas if the base pairing is perfect then RISC will create a break in the mRNA which leads to its rapid degradation.

Dicer is a protein which is essential for the maturation of both miRNA and siRNA, cleaving the double stranded immature forms of these regulators. Consequently, to test whether or not TING is likely to be regulated by miRNA or siRNA, one should assess TING mRNA stability in WT cells and in cells in which Dicer has been knocked down using an exogenous Dicer-specific siRNA. Dicer knockdown would lead to a significant reduction in miRNA/siRNA maturation, meaning that the stability of TING mRNA would be increased if it were regulated by one of these two molecules. TING mRNA stability could be assessed by using a luciferase reporter with the 3' UTR of TING mRNA to visualize stability in the WT and Dicer-knockdown cells. Drosha is a protein essential to the maturation of miRNA but not siRNA, and as such this experiment could be repeated using Drosha-knockdown cells to determine whether an miRNA or an siRNA were likely to be regulating TING mRNA stability.

The miRNA likely to target TING mRNA can be identified using an algorithm which compares the TING 3' UTR sequence to all miRNA sequences and predicts which miRNA are likely to target TING. Once a tentative miRNA has been identified in this way, this result can be confirmed using the abovementioned reporter to assess levels of TING mRNA in WT cells and in cells in which that particular miRNA has been inactivated by site-directed mutagenesis. If TING mRNA levels are higher in the miRNA-/- cells than in the WT cells then this miRNA is likely to regulate TING mRNA stability in vivo.

Acyl-Transferases

The transmembrane acyl-transferase most likely catalyzes the transfer of fatty acid chains to lyso-phospholipids (LPLs), thereby generating phospholipids (PLs) of various shapes. Certain PL conformations favor membrane curvature, and thus if an acyl-transferase responsible for converting a curvature-inducing lipid such as lyso-phosphatidylcholine (LPC) to the planar-favoring PC were inactive, the membrane could be prone to curvature. This excess curvature could cause the membrane to fragment into multiple segments, as seen in this screen. The identified poly-topic integral membrane protein could be an ATP-dependant PL flippase which mediates the translocation of specific amphipathic PLs across the Golgi membrane so as to maintain an asymmetric lipid distribution. This asymmetric distribution is important for generating membrane curvature and is essential for the proper localization of certain proteins integral to membrane stability such as those which bind phosphatidlyserine (PS), a PL which is normally localized to the cytosolic leaflet of the Golgi membrane. The PS-binding protein identified in this screen could be an adaptor protein involved in coated vesicle/tubule formation. Coat adaptors often use PLs to localize only to appropriate membrane surfaces, and a defect in this particular adaptor may have eliminated formation of particular coated vesicles which may be necessary for the induction of membrane curvature, which is an essential function of any vesicle-forming coat protein.

To test whether the poly-topic integral membrane protein (Pmp) is an ATP-dependent phospholipid flippase, one can use a protocol adapted from Natarajan et al. Briefly, Golgi membrane are isolated by ultracentrifugation from WT or Pmp-ts cells in which Pmp activity is normal at 27oC but disrupted at 37oC, leading to Golgi fragmentation. Small amounts of different phospholipids labeled with the fluor NBD should be added to these membranes in the absence of ATP, such that these lipids will slowly distribute between the two membrane leaflets. ATP should then be added to the membranes, and after a period of time the relative distribution of the phospholipid for each membrane (WT, Pmp-27, Pmp-37) should be established and compared to membrane to which ATP was not added. This distribution can be measured by fluorescent reading of samples in which BSA has been used to extract cytosolic but not lumenal NBD-phospholipids. If the distribution of a particular phospholipid is significantly different in WT and Pmp-27 membrane relative to Pmp-37 membrane then this would suggest that Pmp has an ATP-dependent flippase activity which is inactive at 37oC, and this inactivation leads to a lack of Golgi membrane integrity due to improper phospholipid distribution.

Potassium Channels

As long as K+ channels remain open, K+ molecules from within the cell move through these channels down a [K+] gradient. This K+ efflux generates charge and causes the membrane to become hyperpolarized. When the K+ channel closes, K+ efflux is blocked and the membrane rapidly depolarizes, and this depolarization triggers the opening of these voltage-gated Ca2+ channels which remain closed when the membrane is hyperpolarized. If the extracellular K+ concentration was acutely increased to the same level as intracellular [K+] then there would be no [K+] gradient and hence no K+ efflux from the cell. Because K+ ions would not move down a gradient through the K+ channels, no membrane polarity would be generated and the membrane would depolarize just as if the K+ channels were closed. This depolarization would cause the voltage-gated Ca2+ channels to open, leading to subsequent insulin release which would persist until the extracellular [K+] fell below the intracellular [K+] and the membrane once again became hyperpolarized.

If a patient had a mutation in this K+ channel which effectively locked the channel in an open position, then these cells would not be able to release insulin. The constant open state of this channel would ensure that the membrane of these β cells remains hyperpolarized, and hence the voltage-gated Ca2+ channels will remain closed. Because calcium channels do not open, cells will not release insulin and as a result patients with these mutations will present with a form of diabetes which requires insulin therapy. This mutation would likely be dominant, as if even half of a patient's K+ channels remained locked in an open state then the membrane would remain hyperpolarized in the presence of high levels of intracellular ATP because there would still be a constant K+ efflux, leading to persistent closure of the Ca2+ channels. If a patient had a mutation which locked the K+ channel in a closed state then they would constitutively release insulin regardless of glucose levels because their β cell membranes would remain depolarized, resulting in constitutive opening of the voltage-gated Ca2+ channels and causing hyperinsulinemia. This mutation would likely be recessive, as if half of a patient's K+ were able to open and close in response to intracellular ATP then they would still be able to generate a membrane potential and they would thus not have constitutively open Ca2+ channels.

A drug which functions as a transient inhibitor of these K+ channels could be designed so as to stimulate insulin release in Type II diabetic patients. Such a drug would block the K+ channel, leading to membrane depolarization and insulin release. It would be critical that this drug not have a long biological half-life or that it allow limited K+ channel opening, as prolonged closure of the K+ channels would result in deleterious excess insulin secretion.

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