Please answer last two questions o ated by these myosin molecules are presumed t
ID: 98624 • Letter: P
Question
Please answer last two questions o ated by these myosin molecules are presumed to pall the bulk of the | cell along behind the advancing lamellipodum Myosin l a over a 9.15 EXPERIMENTAL PATHWAY Studying Actin-Based Motility without Cells The leading edge of a motile cell is a complex, crowded, and dynamic compartment, Eled with a multitude of dferent types tails" of short randomly oriented actin appeared to have extended from cells, some Listeria Portnoy an crowded, and with long "tais of have extended from one pole of the Listeria IFIGURE 1b). At the ediges ofi to have pushed Singer-like proteins Parsing out the roles of each of these proteins great interest to cell biologists, but dfficult to accomplish withn the context of a living cell. It is for this reason that for many years research has focused on a simplified and pared-down system of these proteins is of then a cometike appearance t e easoh for many years, sions outwand from the cell membrane. Based on these of the Gram-positive were used to propel Listeria through the cytoplasm, and wet taly d Tiney postulated in 1989 that the actin The description of the comet tails behind Listenia Thenot, a gradate student n the laboratory of Tim Mit- that the actin comet pared down system for cell motility-intracellular movement of the Gram-postive mately used to gain aof the comet tal were used to press to a new host cell. bacterium Listenia monocytogenes. The discovery and refinemente of this model system has been an important experimental tool that has greatly augment works are used to generate force in vivo. to gain access to a behind Listeria r drg of how actin net intrgang to cell to log sts studying hea tr cytoskeleton. ol was fist descrbedin t the University of California, San Francisco, sow parslieitre ween her studies of actin filament dynamics in locoms b report in 1926 by E.G.D. Murray and colleagues, who were studyod the postulated ing the cause of an epidemic observed in rabbits and guinea eibits and guinea and the postulated propulsion of Listeria cells in the n cal ums,but it was not until During the course of her graduate studies, Theriot de agues, who were study ing the cause of an epidemic pigs. Listeriosis was later observed in humans, but it was not until the early 1980s that listeriosis was linked to the ingestion of con- Theriot described for the first time, the dynamics of actin networks in living loca moting cells using fuorescently tagged actin and a homamaoe taminated food products. Foodborne illness caused by Listeria caused by Listeria can sometimes be severe, particu/arly in immunocompromised video microscopy system. To follow a specitic population individuals, but it more often causes fever and gastrointestinal actin monomers over time. Theriot coupied a photoactivatable fluorescent compound to actin monomers and labeled actin into live cells. She then allowed over time, Theriot coupled a photoactivatable actin into live cells. She then allowed the labeled actin to symptoms in healthy adults Research into the cause of lsteriosis began in earmest in the mid-1980%, leading multiple labs to discover that Listeria could be incorporated into actin polymers in the cell. By shining light enter and multiply within the cytosol of clls, Interestingly, some a specific wavelength in a small rectangular-shaped region of the researchers, including Ed Havell at the Trudeau Institute and Danel amellipod, Theriot could turn on the fluorescence of the labeled Portnoy at Washington University, observed that the infection could actin and observe how the labeled filamentous actin behaved spread from one cell to its neighboring cels on a petri dish even in t le cells Figure 1 a) By activating a reg on at the tof the presence of extracelular antibiotic, suggesting that the the lamellipod, she observed that the labeled actin flaments bacteria were somehow able to infect new cells directly from their remained organized and stationary relative to the substrate as previous host cell.3- Portnoy suspected that the mode of infectionthe cell moved forward. This was the case for all of the craw. might be actin-based from his observations and that the addition ing cells she observed, no matter how quickly they moved acrom of cytochalasin, an inhibitor of actin polymerization, could block the the substrate. The bar of fluorescence eventually dispersed and bacteria from spreading to new cells. To get a better view of what disappeared as the bar approached the rear of the cell, sugges Listeria was doing in the cell cytosol, Portnoy, then at the University ing that filaments were eventually completely dismantled and of Pennsylvania, began a collaboration with Lew Tiney, a prominent the monomers recycled. From these observations, Theriot and electron microscopist. Using a macrophage-lke cell ine, Portnoy Mitchison proposed that extension of the lamellipodium during infected one set of petri dishes with wild-type Listeris, and, as a cell motility was caused by the polymerization of new actin la negative control, infected another set with a Listeria mutant that ments at the leading edge (rather than from the rearrangement was unable to enter cells. At specific timepoints, the cells were fixed and prepared for electron microscopy. Two hours after infection, of existing filaments). plasm were surrounded by a cloud of actin, which was confirmed the movement of Listeria. In this through the decoration of the filaments with myosin $1 fragments. tivatable actin mon At the four hour timepoint, numerous bacteria were observed Listeria and selected actin t in collsaboration with Portnoy and Tilney, Theriot and Mitchison en utlized the experimental system they developed to study t case. Theriot injected phohos through the decoration of the filaments with myosin Si fragments. tivatable actin monomers into cells that had been infected wit a and selected actin tails to study in more detail uing ails to study in more detail usngExplanation / Answer
1. How ActA causes nucleation of actin protein
ActA is not directly involved in the nucleation of actin protein. ActA while on the surface of Listeria, transiently interact with Arp 2/3 and stimulates nucleation activity of Arp2/3. Arp2/3 complex then acts as a template for a new actin filament and gets dissociated from ActA and gets incorporated into the growing actin tail.
2. Two experiments that helped draw conclusions about ActA
Expt 1: “Listeria mutants that lacked ActA were shown to be unable to recruit actin filaments and no longer showed motility in infected cells.”
Expt 2: “Expression of ActA in previously non-motile strains of bacteria allowed for the formation actin cornet tails and motility characteristic of pathogenic strains.”