The journal of cell biology format

Essay Instruction

Your essay must include a title page, in-text references and a references page in aIDition to the four pages of text. Four of these references must be literature that is not cited in your original paper, and these citations must be highlighted in your final version. Formatting is important! We will follow the Journal of Cell Biology format which includes citations from one or two authors as (Smith and Williams, 2007). Citations from more than two authors would be cited as (Smith et al., 2007). For your references page, you can follow the format of the citation below. 

Bear, J.E., T.M. Svitkina, M. Krause, D.A. Schafer, J.J. Loureiro, G.A. Strasser, I.V. Maly, O.Y. Chaga, J.A. Cooper, G.G. Borisy, and F.B Gertler. 2002. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell. 109: 509-521. 

The outline of your essay should be as follows:

                1. Title Page

                2. Introduction/Background (with citations)

                3. Results and Findings of the paper

                4. New Questions

                5. Experimental Approaches to AIDress New Questions

                6. References 

You should spend about half of your paper aIDressing the introduction and results, and the other half aIDressing the new questions and proposed experiments.

 Student Name

UID 111111111 

Reference Paper: 

Hagedorn E.J., J.W. Ziel, M.A. Morrissey, L.M. Linden, Z. Wang, Q. Chi, S.A. Johnson, D.R. Sherwood. 2013. The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo. Journal of Cell Biology.201(6): 903-913. 

Introduction:UNC-6 (netrin) has been shown to signal protrusive activity in the growth cone of axons by interacting with UNC-40 (netrin receptor) inC. elegans (Norris and Lundquist, 2011). In C. elegans,netrin is produced by the ventral nerve cord, and has been found to promote anchor cell invasion into vulval tissue(Ziel and Sherwood, 2010). In neurons, UNC-40 reacts to the netrin ligand by the creation of a UNC-40 homodimer,(Norris and Lundquist, 2011).Known effectors of UNC-40 include UNC-34 and MIG-2 (Hagedorn et al. 2013). UNC-34 encodes the enabled/ VASP ortholog protein and is known to affect actin organization (Shakir, et al., 2006).MIG-2 is has been shown to be required for C.elegansneuroblast migration and protrusion (Dyer et al., 2010).MIG-2 is a RacGTPase that along with UNC-34 functions to control actin based protrusive structures (Shakir et al., 2006).In neuron migration UNC-34 and MIG-2 have overlapping roles, and UNC-34 acts in parallel to CED-10, MIG-2, and RAC-2 (Shakir et al., 2006).

            In order to understand how cells breach and cross the basement membrane during normal developmental process and cancer metastasis in vivo, Hagedorn et. al used anchor cells in C. elegans as a model. In C. elegans, dynamic, F-actin rich, high turnover invadopodiabreach the basement membrane giving rise to a stable invasive protrusion that results in displacement of the basement membrane, allowing the anchor cells to migrate into vulval tissue (Hagedorn et al., 2013).While attempting to understand the molecular mechanism behind the transition from the invadopodia to the invasive protrusion in anchor cells, Hagedorn et al. found that UNC-40 localizes to the site of the membrane breach, becomes activated by UNC-6, andrecruits effector proteins (UNC-34, MIG-2) that regulate cytoskeletal formation and are required for the stable invasive protrusion to form. They also found that UNC-40 is required for physical displacement of the basement membrane at the breach site.

Results: Hagedorn et al. visualized, using fluorescent microscopy, that the AC crosses the basement membrane using in vivo invadopodia. F-actin was visualized using a cdh-3 >mcherry::moeABDprobe. It was found that the F-actin foci depress into the basement membrane prior to breaching, are highly dynamic, and localize to sites of basement membrane breach. These results are similar to known characteristics of invadopodia,suggestingthat the AC is breaching the basement membrane using in vivo invadopodia.

            Next,Hagedorn et al. found that the letrin receptor, UNC-40 (DCC), localizes to the site of the basement membrane breach approximately 20 minutes before the initial breach. The investigators constructed a UNC-40::GFP fusion protein in order to visualize UNC-40 with fluorescent microscopy. A UNC-40 knockout did not restrict the AC’s ability to form invadopodia, but the initial breach was delayed by 1.5 hrs. It was also found that in unc-40 (e271) mutants there were multiple invasive protrusions formed instead of a single invasive protrusion, and the rate at which the breach widened in these mice was reduced by half. These results suggest that the UNC-40 functionsto create an opening in the basement membrane and the formation of the invasive protrusion. They also found that unc-6 (ev400) mutants have a similar, but not as severe phenotype as unc-40 (e271) mutants, which along with previous research suggests that UNC-40 function requires activation by UNC-6. 

            Hagedorn et al., next aIDressed how UNC-40 causes the formation of the stable invasive protrusion. UNC-40 effectors were labeled by constructing UNC-34::GFP and MIG-2::GFP fusion proteins.  In wild type cells, these effectors were found to localize to the site of the breach in the basement membrane where UNC-40 localized. The investigators then tested loss of function of UNC-40 using a unc-40(e271) mutant, and found that localization of UNC-34::GFP and MIG-2::GFP was lost. In a separate experiment it was found that loss of function mutations of UNC-34::GFP and MIG-2::GFP resulted in a complete loss of invasive protrusion. These results suggest that UNC-40 recruits the effector proteins UNC-34 and MIG-2 to the site of the basement membrane breach, and that these effectors are required for the invadopodia to invasive protrusion transition.

            Hagedorn et al., also found that labeled ECM beneath the cell accumulates at the edge of the breach of the basement membranesuggesting that the basement membrane is being physically displaced.They also found that UNC-40 is required for this physical displacement of the basement membrane, as a mutant loss of function UNC-40 (e271) resulted in a loss of accumulation of the basement membrane. 

New Questions:

–       Does expression of constitutively active UNC-40 homodimers still lead to the transition from invadopodia to an invasive protrusion in the absence of UNC-6?

–       Does overexpression of UNC-40 affect the transition of the invadopodia to the invasive protrusion?

–       Due to the redundant effects of UNC-34 and MIG-2 is UNC-34 required for the transition from an invadopodia to an invasive protrusion?

Experimental Approaches to New Questions:It has been shown in vertebrates that UNC-40 forms a homo dimer in response to netrin(Norris and Lundquist, 2011). In anchor cells if you express mutant UNC-40 proteins that have a high affinity to homo-dimerize, and knockdown UNC-6it is possible to test if a constitutively active mutant UNC-40 can function in the absence of UNC-6. First, it is necessary to createone strain of C.eleganswith a mutant UNC-40 gene that has a high affinity to homo-dimerize, along with a cell membrane marker cdh-3> GFP::CAAX, which is used to visualize the cells under fluorescent microscopy; and a second strain with only the cell marker. First, it must be tested to make sure that the mutant UNC-40protein is still functional, which can be done using a fluorescent microscope and observing cell migration. If the protein is functional and wildtype phenotype is observed, it then must be tested to see if the protein forms a dimer in the absence of UNC-6. This is done by using a UNC-6 MO to knockdown UNC-6 in the mutant and control, and then extracting cell lysate and precipitating UNC-40 using an anti UNC-40 ab, and running on a western blot, using a labeled anti-UNC-40 ab to visualize the proteins. The control with wildtype UNC-40 and UNC-6 MO should be used and run on the same gel in a separate lane. The band should move slower on the gel with the mutant protein when compared to the control, as it is predicted that the mutant will form a dimer in the absence of UNC-6. It is also necessary to compare the phenotype of the mutant UNC-40 in the presence of UNC-6 MO to the phenotype of wildtype UNC-40 in the presence of UNC-6 MO. The results are expected to show that the wildtype UNC-40 fails to form an invasive protrusion in the absence of UNC-6, while the mutant UNC-40 remains able to form the invasive protrusion transition. These results would suggest that UNC-6 activates the UNC-40 gene, and a mutant form of UNC-40 that is constitutively active does not require UNC-6 in order to be activated.

The results presented by Hagedorn et al. show that UNC-40 is required for the invadopodia to the invasive protrusion transition, but do not aIDress whether or not overexpression of UNC-40 can affect the invadopodia to invasive protrusion transition. First, a UNC-40::GFP, laminin::cherry transgene, and a cell membrane marker cdh-3> GFP::CAAX transgene are expressed in anchor cells. Two groups of anchor cells can be used, one which includes exogenous UNC-40::GFP mRNA injected into the cell, and the other a control, in which just the vehicle is injected into the cell. Using fluorescent microscopy one can measure the amount of UNC-40::GFP present in both groups of anchor cells. Groups with exogenous UNC-40::GFP mRNA should have more protein present, and fluorescence should be increased. Using fluorescence microscopy visualization of the transition of the invadopodia to the invasive protrusion by the cell can be visualized using the GFP::CAAX cell membrane marker. The size of the basement membrane breach can also be visualized by using fluorescent microscopy to visualize the displacement of the laminin::cherry in the basement membrane. Using video microscopy, one can measure the average rate of the hole expansion, as well as the final size of the breach in the basement membrane of both the control group treated with vehicle and the group treated with exogenous UNC-40::GFP mRNA.Gain of function UNC-40::GFP may create localization of UNC-40 to sitesother than basement membrane breach and result in multiple invasive protrusions, but another result may occur in which this gain of function results in a more distinct breach and quicker invadopodia to invasive protrusion transition.

A unc-34(e951) and mig-2(mu28) double mutant created a complete loss of phenotype; however, due to the redundant effects of MIG-2 and UNC-34 in axon migration described by Shakir et al., it is possible that UNC-34 is not required to form an invasive protrusion if MIG-2 is functional. This can be tested by using a single unc-34(e951) mutant and identifying the movement of the anchor cell through the basement membrane using fluorescent microscopy, and using an AC specific PI(4,5)P2 probe to visualize the anchor cell. If UNC-34 is required for the invasive protrusion to form, then the invasive protrusion will not be visualized, the AC will not migrate across the basement membrane, and it is probable that there will be multiple invadopodia, and a phenotype similar to the double mutant is expected, although not as severe. The control would consist of visualizing a non-mutant AC, using the same probe as before, and the invadopodia should transition into an invasive protrusion and cell migration should occur.

References:

Dyer, J.O., and R.S. Demarco, and E.A. Lundquist. 2010. Distinct roles of RacGTPases and the UNC-73/Trio and PIX-1 Rac GTP exchange factors in neuroblast protrusion and migration in C. elegans. Small GTPases. 1(1):44-61.

Hagedorn E.J., J.W. Ziel, M.A. Morrissey, L.M. Linden, Z. Wang, Q. Chi, S.A. Johnson, D.R. Sherwood. 2013. The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo. Journal of Cell Biology.201(6): 903-913.

Norris, A.D., and E.A. Lundquist. 2011. UNC-6/netrin and its receptors UNC-5 and UNC-40 DCC modulate growth cone protrusion in vivo in C. elegans. Development.138(20): 4433-4442.

Shakir, M.A., J.S. Gill, and E.A. Lundquist. 2006. Interactions of UNC-34 Enabled with GTPases and the NIK kinase MIG-15 in Caenorhabditiselegans axon pathfinding and neuronal migration. Genetics. 172(2): 893-913.

Ziel J.W., and D.R. Sherwood. 2010. Roles for netrin signaling outside of axon guidance: a view from the worm. Developmental Dynamics. 239(5):1296-1305.

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