The Radish Gene Reveals a Memory Component with Variable Temporal Properties

Memory phases, dependent on different neural and molecular mechanisms, strongly influence memory performance. Our understanding, however, of how memory phases interact is far from complete. In Drosophila, aversive olfactory learning is thought to progress from short-term through long-term memory phases. Another memory phase termed anesthesia resistant memory, dependent on the radish gene, influences memory hours after aversive olfactory learning. How does the radish-dependent phase influence memory performance in different tasks? It is found that the radish memory component does not scale with the stability of several memory traces, indicating a specific recruitment of this component to influence different memories, even within minutes of learning

Reconstituting the Cvt pathway: An approach to unraveling autophagy [abstract]

Faculty Mentor: Dr. Silvia Jurisson, RadiochemistryAbstract only availableAutophagy, literally self-cannibalism, is a highly regulated catabolic process that is important in the maintenance of all eukaryotic cells. In humans, both excess and insufficient autophagy is linked to many diseases including cancer, Huntington's and Parkinson's diseases. Autophagy is the process in which defective and unwanted organelles along with cytoplasm are taken into an autophagosome and transported to the vacuole or lysosome to be degraded and used to build new macromolecules. Although most of the proteins involved in the autophagic pathway are known, their specific functions remain unknown. In order to better understand the molecular mechanisms of autophagy, we are using a specialized autophagic pathway found only in the yeast Saccharomyces cerevisiae: the Cytoplasm to vacuole targeting (Cvt) pathway. The Cvt pathway is used to deliver the inactive precursor of aminopeptidase1 (prApe1), to the vacuole. The pathway begins in the cytosol when prApe1 aggregates into dodecamers, which then form the Ape1 complex. This is followed by Atg19 binding to the Ape1 complex. Atg19 interacts with Atg11 (and possibly Atg8), which recruits autophagic membrane. These proteins along with a few others complete the Cvt vesicle, which then fuses with vacuolar membrane allowing Ape1 to be released into the lumen of the vacuole. We are attempting two different approaches to understand the Cvt pathway. In our first approach we are trying to reconstitute the Cvt pathway in Pichia pastoris, a related yeast strain in which the pathway does not occur. So far we have been successful in expressing S. cerevisiae's ape1 and atg19 genes in P. pastoris. In these strains the Ape1 complex aggregates and Atg19 co-localizes. We are still working on expressing atg11 in P. pastoris. Our second approach is to form the Cvt vesicle in vitro. In S. cerevisiae stable Ape1 complexes can be observed microscopically. Ape1 is also stable when co-localized with Atg19 as seen in the microscope. We are currently trying to purify the Ape1 complex, which is essential for using as a scaffold for in vitro formation of the Cvt vesicle

Reconstituting the Cvt pathway in pichia pastoris [abstract]

Abstract only availableIn yeast, autophagy is primarily a response to nutritional stress in which the cell sacrifices some of its cytoplasm and organelles in order to survive. Saccharomyces cerevisiae contains a specialized pathway for the delivery of certain proteins to the vacuole by selective autophagy called the Cytoplasm to vacuole targeting (Cvt) pathway. While autophagy is generally believed to be non-selective, the Cvt pathway is highly specific, transporting only a few selected proteins to the vacuole. The two known cargo proteins of the Cvt pathway are -mannosidase1 (Ams1) and the inactive precursor of aminopeptidase1 (prApe1). The pathway begins in the cytosol when prApe1 oligomerizes into dodecamers, which then aggregate to form the Ape1 complex. We are using the Ape1 aggregate as a model system for studying how protein aggregates are delivered to the vacuole/lysosome by constitutive and selective autophagy. We have introduced Ape1 from S. cerevisiae into the yeast Pichia pastoris in an attempt to reconstitute the Cvt pathway in this yeast. P. pastoris is related to S. cerevisiae, but does not contain the Cvt pathway. We are currently studying the requirements for the uptake of Ape1 into the vacuole in P. pastoris.McNair Scholars Progra

Mutation of the <i>rsh</i> gene does not influence conditioning or place memory tested directly after training.

<p>Following a 30 s pre-test period (black bars), wild-type CS and <i>rsh¹</i> mutant flies were trained in two equal length periods for a total of either 6 or 20 min with 41°C (light gray bars). A 3 min memory was tested directly following in the post-test period (dark gray bars). The training, retention intervals, and testing patterns (both pre and post) are diagrammed for each panel, the time axis is not to scale. (<i>A</i>), Conditioning and memory tests were similar between the genotypes with 6 min of training (N = 331; pre-test: U = 12753.5, z = 1.07, P = 0.28; 1^st training period: U = 11877.0, z = 2.08, P = 0.04; 2^nd training period: U = 12888.5, z = 0.92, P = 0.36; post-test: U = 13237.0, z = 0.51, P = 0.61). (<i>B</i>) Conditioning and memory tests were also similar between the genotypes with 20 min of training (N = 232; pre-test: U = 6106.5, z = 1.22, P = 0.22; 1^st training period: U = 5740.5, z = 1.93, P = 0.06; 2^nd training period: U = 5802.0, z = −1.81,  = 0.07; post-test: U = 6463.0, z = −0.52, P = 0.60). (<i>C</i>) The <i>rsh</i> gene is necessary for normal short-term place memory. Flies were trained with intermittent training and then held for varying times (1 – 40 min) before being tested for memory with a short reminder training. The <i>rsh¹</i> flies had memory performance similar to wild-type CS levels with a 1 min delay between training and the memory test (N = 447, U = 24641.5, z = 0.24, P = 0.8). Significant differences were found at several time points following training (10 min: N = 295, U = 8637.0, z = .02, **  =  P<0.01; 20 min: N = 330, U = 10074.5, z = 3.95, ***  =  P<0.001; 30 min: N = 311, U = 10926.0, z = 1.45, P = 0.1; 40 min: N = 351, U = 12941.5, z = 2.48, **  =  P<0.01). The values are means and error bars represent s.e.m.</p

Control behaviors of wild-type CS and <i>rsh¹</i> mutant flies.

<p>MCH avoidance: ANOVA F(3,32)  = 1.07, P = 0.4; Oct avoidance: F(3,20)  = 1.3, P = 0.3; Sugar attractiveness: ANOVA F(3,44) = 0.75, P = 0.53; Activity: F(1,561) = 3.3, P = 0.07.</p

Mutation of the <i>rsh</i> gene reveals a major role in aversive olfactory memory (ARM) and is necessary for appetitive olfactory memory shortly after conditioning.

<p>Flies were either trained with odorants paired with electric shock or sugar reward. The training, cold-shock, retention intervals, and testing patterns (both pre and post) are diagrammed for each panel, the time axis is not to scale. (<i>A</i>) Olfactory memory tested three min after training is reduced in <i>rsh¹</i> flies compared to CS flies, although levels do not reach statistical significance (F(1,12)  = 3.5, P = 0.09). To reveal the <i>rsh</i> function in aversive olfactory memory, wild-type CS and <i>rsh¹</i> flies were trained with odorant / shock pairings, then after 2 hrs were given a cold-shock, memory was tested 1 hr later. Memory performance of <i>rsh¹</i> flies was significantly lower than wild-type CS flies with this procedure (F(1,10)  = 5.0, *  =  P = 0.04). (<i>B</i>) Appetitive olfactory short-term memory was tested at 3, 30, and 60 min after the odorant / sucrose training session. A <i>rsh¹</i> phenotype was evident at all tested time points after training (3 min: F(1,16)  = 29.2, ***  =  P<0.001; 30 min: F(1,14)  = 12.3, **  =  P<0.01; 60 min: F(1,14)  = 12.1, **  =  P<0.01). (<i>C</i>) The <i>rsh¹</i> appetitive short term olfactory memory phenotype is rescued with a transgenic copy of the wild-type version of the <i>rsh</i> gene (F(3,32)  = 13.0, P<0.0001; post-hoc tests: CS vs <i>rsh¹</i> ***  = P<0.001, <i>rsh¹</i> vs. <i>rsh¹</i>; hs-<i>rsh-1</i> *  = P<0.05, CS vs. <i>rsh¹</i>; hs-<i>rsh-1</i>, *  = P<0.05; <i>rsh¹</i> vs. CS; hs-<i>rsh-1</i> *  = P<0.05; CS vs. CS; hs-<i>rsh-1</i> *  = P<0.05). The values are means and error bars represent s.e.m.</p