Two adaptation mechanisms regulate cellular migration in Dictyostelium discoideum

Dictyostelium discoideum is a simple model widely used to study many cellular functions, including differentiation, gene regulation, cellular trafficking and directional migration. Adaptation mechanisms are essential in the regulation of these cellular processes. The misregulation of adaptation components often results in persistent activation of signaling pathways and aberrant cellular responses. Studying adaptation mechanisms regulating cellular migration will be crucial in the treatment of many pathological conditions in which motility plays a central role, such as tumor metastasis and acute inflammation. I will describe two adaptation mechanisms regulating directional migration in Dictyostelium cells. The Extracellular signal Regulated Kinase 2 (ERK2) plays an essential role in Dictyostelium cellular migration. ERK2 stimulates intracellular cAMP accumulation in chemotaxing cells. Aberrant ERK2 regulation results in aberrant cAMP levels and defective directional migration. The MAP Phosphatase with Leucine-rich repeats (MPL1) is crucial for ERK2 adaptation. Cells lacking, MPL1 (mpl1- cells) displayed higher pre-stimulus and persistent post-stimulus ERK2 phosphorylation, defective cAMP production and reduced cellular migration. Reintroduction of a full length Mpl1 into mpl1- cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild type cells (Wt). These results suggest Mpl1 is essential for proper regulation of ERK2 phosphorylation and optimal motility in Dictyostelium cells. Cellular polarization in Dictyostelium cells in part is regulated by the activation of the AGC-related kinase Protein Kinase Related B1 (PKBR1). The PP2A regulatory subunit, B56, and the Glycogen Synthase Kinase 3 (GSK3) are necessary for PKBR1 adaptation in Dictyostelium cells. Cells lacking B56, psrA-cells, exhibited high basal and post-stimulus persistent phosphorylation of PKBR1, increased phosphorylation of PKBR1 substrates, and aberrant motility. PKBR1 adaptation is also regulated by the GSK3. When the levels of active GSK3 are reduced in Wt and psrA- cells, high basal levels of phosphorylated PKBR1 were observed, in a Ras dependent, but B56 independent mechanism. Altogether, PKBR1 adaptation is regulated by at least two independent mechanisms: one by GSK3 and another by PP2A/B56.

Two Adaptation Mechanisms Regulate Cellular Migration in Dictyostelium discouideum

Dictyostelium discoideum is a simple model widely used to study many cellular functions, including differentiation, gene regulation, cellular trafficking and directional migration. Adaptation mechanisms are essential in the regulation of these cellular processes. The misregulation of adaptation components often results in persistent activation of signaling pathways and aberrant cellular responses. Studying adaptation mechanisms regulating cellular migration will be crucial in the treatment of many pathological conditions in which motility plays a central role, such as tumor metastasis and acute inflammation. I will describe two adaptation mechanisms regulating directional migration in Dictyostelium cells. The Extracellular signal Regulated Kinase 2 (ERK2) plays an essential role in Dictyostelium cellular migration. ERK2 stimulates intracellular cAMP accumulation in chemotaxing cells. Aberrant ERK2 regulation results in aberrant cAMP levels and defective directional migration. The MAP Phosphatase with Leucine-rich repeats (MPL1) is crucial for ERK2 adaptation. Cells lacking, MPL1 (mpl1- cells) displayed higher pre-stimulus and persistent post-stimulus ERK2 phosphorylation, defective cAMP production and reduced cellular migration. Reintroduction of a full length Mpl1 into mpl1- cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild type cells (Wt). These results suggest Mpl1 is essential for proper regulation of ERK2 phosphorylation and optimal motility in Dictyostelium cells. Cellular polarization in Dictyostelium cells in part is regulated by the activation of the AGC-related kinase Protein Kinase Related B1 (PKBR1). The PP2A regulatory subunit, B56, and the Glycogen Synthase Kinase 3 (GSK3) are necessary for PKBR1 adaptation in Dictyostelium cells. Cells lacking B56, psrA-cells, exhibited high basal and post-stimulus persistent phosphorylation of PKBR1, increased phosphorylation of PKBR1 substrates, and aberrant motility. PKBR1 adaptation is also regulated by the GSK3. When the levels of active GSK3 are reduced in Wt and psrA- cells, high basal levels of phosphorylated PKBR1 were observed, in a Ras dependent, but B56 independent mechanism. Altogether, PKBR1 adaptation is regulated by at least two independent mechanisms: one by GSK3 and another by PP2A/B56.

Implicit eyewitness memory

After a crime has occurred, one of the most pressing objectives for investigators is to identify and interview any eyewitness that can provide information about the crime. Depending on his or her training, the investigative interviewer will use (to varying degrees) mostly yes/no questions, some cued and multiple-choice questions, with few open-ended questions. When the witness cannot generate any more details about the crime, one assumes the eyewitness' memory for the critical event has been exhausted. However, given what we know about memory, is this a safe assumption? In line with the extant literature on human cognition, if one assumes (a) an eyewitness has more available memories of the crime than he or she has accessible and (b) only explicit probes have been used to elicit information, then one can argue this eyewitness may still be able to provide additional information via implicit memory tests. In accordance with these notions, the present study had two goals: demonstrate that (1) eyewitnesses can reveal memory implicitly for a detail-rich event and (2) particularly for brief crimes, eyewitnesses can reveal memory for event details implicitly that were inaccessible when probed for explicitly. Undergraduates (N = 227) participated in a psychological experiment in exchange for research credit. Participants were presented with one of three stimulus videos (brief crime vs. long crime vs. irrelevant video). Then, participants either completed a series of implicit memory tasks or worked on a puzzle for 5 minutes. Lastly, participants were interviewed explicitly about the previous video via free recall and recognition tasks. Findings indicated that participants who viewed the brief crime provided significantly more crime-related details implicitly than those who viewed the long crime. The data also showed participants who viewed the long crime provided marginally more accurate details during free recall than participants who viewed the brief crime. Furthermore, participants who completed the implicit memory tasks provided significantly less accurate information during the explicit interview than participants who were not given implicit memory tasks. This study was the first to investigate implicit memory for eyewitnesses of a crime. To determine its applied value, additional empirical work is required.

Implicit Eyewitness Memory

After a crime has occurred, one of the most pressing objectives for investigators is to identify and interview any eyewitness that can provide information about the crime. Depending on his or her training, the investigative interviewer will use (to varying degrees) mostly yes/no questions, some cued and multiple-choice questions, with few open-ended questions. When the witness cannot generate any more details about the crime, one assumes the eyewitness’ memory for the critical event has been exhausted. However, given what we know about memory, is this a safe assumption? In line with the extant literature on human cognition, if one assumes (a) an eyewitness has more available memories of the crime than he or she has accessible and (b) only explicit probes have been used to elicit information, then one can argue this eyewitness may still be able to provide additional information via implicit memory tests. In accordance with these notions, the present study had two goals: demonstrate that (1) eyewitnesses can reveal memory implicitly for a detail-rich event and (2) particularly for brief crimes, eyewitnesses can reveal memory for event details implicitly that were inaccessible when probed for explicitly. Undergraduates (N = 227) participated in a psychological experiment in exchange for research credit. Participants were presented with one of three stimulus videos (brief crime vs. long crime vs. irrelevant video). Then, participants either completed a series of implicit memory tasks or worked on a puzzle for 5 minutes. Lastly, participants were interviewed explicitly about the previous video via free recall and recognition tasks. Findings indicated that participants who viewed the brief crime provided significantly more crime-related details implicitly than those who viewed the long crime. The data also showed participants who viewed the long crime provided marginally more accurate details during free recall than participants who viewed the brief crime. Furthermore, participants who completed the implicit memory tasks provided significantly less accurate information during the explicit interview than participants who were not given implicit memory tasks. This study was the first to investigate implicit memory for eyewitnesses of a crime. To determine its applied value, additional empirical work is required.

Specific binding of the mammalian high mobility group protein AT-hook 2 to the minor groove of AT -rich DNAs: Thermodynamic and specificity studies

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small transcriptional factor involved in cell development and oncogenesis. It contains three "AT-hook" DNA binding domains, which specifically recognize the minor groove of AT-rich DNA sequences. It also has an acidic C-terminal motif. Previous studies showed that HMGA2 mediates all its biological effects through interactions with AT-rich DNA sequences in the promoter regions. In this dissertation, I used a variety of biochemical and biophysical methods to examine the physical properties of HMGA2 and to further investigate HMGA2's interactions with AT-rich DNA sequences. The following are three avenues perused in this study: (1) due to the asymmetrical charge distribution of HMGA2, I have developed a rapid procedure to purify HMGA2 in the milligram range. Preparation of large amounts of HMGA2 makes biophysical studies possible; (2) Since HMGA2 binds to different AT-rich sequences in the promoter regions, I used a combination of isothermal titration calorimetry (ITC) and DNA UV melting experiment to characterize interactions of HMGA2 with poly(dA-dT) 2 and poly(dA)poly(dT). My results demonstrated that (i) each HMGA2 molecule binds to 15 AT bp; (ii) HMGA2 binds to both AT DNAs with very high affinity. However, the binding reaction of HMGA2 to poly(dA-dT) 2 is enthalpy-driven and the binding reaction of HMGA2 with poly(dA)poly(dT) is entropy-driven; (iii) the binding reactions are strongly depended on salt concentrations; (3) Previous studies showed that HMGA2 may have sequence specificity. In this study, I used a PCR-based SELEX procedure to examine the DNA binding specificity of HMGA2. Two consensus sequences for HMGA2 have been identified: 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These consensus sequences have a unique feature: the first five base pairs are AT-rich, the middle four to five base pairs are GC-rich, and the last five to six base pairs are AT-rich. All three segments are critical for high affinity binding. Replacing either one of the AT-rich sequences to a non-AT-rich sequence causes at least 100-fold decrease in the binding affinity. Intriguingly, if the GC-segment is substituted by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step towards finding its binding sites within the genome.