Abstract

Process Oriented Guided Inquiry Learning (POGIL) is an effective approach for group work in STEM courses. POGIL-style lessons foster active learning because they require students to communicate and co-construct knowledge to solve problems. Here, we have designed a POGIL problem set to help students learn about experiments that led to discoveries about protein import into the endoplasmic reticulum (ER). Approximately 30% of all known proteins get imported into a cell’s ER, where they either stay or travel to another location in the secretory pathway. Because this initial localization to the ER is shared amongst all secretory proteins, it is important for cell biology students to understand the mechanism of ER import. Our problem set directs students to answer questions about how an ER import assay works and how the channel for import (known as the translocon) was discovered. This lesson has been used over 100 times at our institution, where students and instructors alike have found it to be successful in solidifying students’ knowledge of ER import and facilitating students’ experimental design skill development.

Primary Image: Protein import into ER: The image shows a protein being inserted into the ER through the translocon.

Citation

Society Learning Goals

Cell Biology

• Protein Targeting & Trafficking
• How are cellular components targeted and distributed to different regions and compartments of a cell?
• Methods & Tools of Cell Biology
• How do the methods and tools of cell biology enable and limit our understanding of the cell?

Science Process Skills

• Process of Science
• Locate, interpret, and evaluate scientific information and primary literature
• Plan, evaluate, and implement scientific investigations
• Interpret, evaluate, and draw conclusions from data
• Communication and Collaboration
• Share ideas, data, and findings with others clearly and accurately

Lesson Learning Goals

• understand how an ER import assay is used.
• understand how a crosslinking experiment is used.
• understand how immunoprecipitation is used.
• value how stepwise experiments can reveal details of cellular mechanisms.
• value how microsomes are useful for ER research.

Lesson Learning Objectives

• interpret data from ER import assays.
• explain how microsomes, protease K, and Triton X are used to measure protein import into the ER.
• design an experiment that will test for signal sequence sufficiency using an ER import assay.
• interpret data from an experiment involving immunoprecipitation (IP).
• explain how crosslinking and immunoprecipitation experiments helped identify translocon proteins.

Introduction

Background

Proteins are constantly being transported to their proper locations in cells. Protein transport is necessary because the roles of proteins are location-specific, meaning proteins cannot function properly unless they are correctly localized. Most protein transport mechanisms, such as the secretory pathway, are conserved (1). The secretory pathway is a series of steps that occur to localize a protein from the site of synthesis to one of the following locations: the endoplasmic reticulum (ER), Golgi, endosome, lysosome, plasma membrane, or extracellular space (2). It is estimated that 30% of all known proteins undergo at least the first step of the secretory pathway, which is import into the ER (2). This step is required for secretory proteins because the ER is where proteins are folded and, if necessary, are integrated into the membrane. Because ER import is vital for proper protein structure and localization, it is important for students to learn about the import mechanism and its discovery. In addition, understanding how we can use purified ER (microsomes) to test whether a protein has been imported is important because it is a common assay carried out in this subfield.

We wrote this lesson to help students learn about two key experiments that have been crucial for the field’s understanding of protein import into the ER: the Dobberstein lab’s ER import assay (3) and the Rapoport lab’s translocation crosslinking experiment (4). We chose to highlight these experiments because they represent how research techniques can be used in innovative ways to learn more about the cell.

In our lesson, students are required to draw on their prior knowledge, such as the function of protease or how to read an autoradiograph, to solve problems. This lesson gives students practice reading experimental descriptions, which helps them develop fluency in distinguishing which aspects of an experiment may have an impact on the outcome. The lesson also requires students to derive meaning from experimental results.

Our lesson differs from the existing ER import CourseSource lesson (5) in the following ways: (i) students will use their knowledge of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to interpret the results of an autoradiograph of a gel, (ii) students will learn about immunoprecipitation and photo-crosslinking and use their knowledge of these techniques to interpret the results of a translocation crosslinking experiment, (iii) students will provide reasoning for various steps performed or conclusions made in these experiments, (iv) students will design their own experiment using the ER import assay, and (v) our lesson follows the Process-Oriented Guided Inquiry Learning (POGIL) approach designed for student learning in small groups (6).

Intended Audience

We designed this lesson for upper level (i.e., junior and senior) life science majors taking a cell biology course. We have taught this lesson to students at a large research university; however, it is designed to be used at any four-year college or university. Students will need to have taken a course such as biochemistry or genetics where they learned about translation, use of antibodies for experiments, and centrifugation. This lesson can be used in a small size classroom (~50 students) or in a breakout session/recitation section of a larger lecture course.

Required Learning Time

This lesson requires 75 minutes: 5 minutes to get students set up in teams, 50 minutes for students to complete the problem set, and 20 minutes to discuss the answers together as a class. The shorter adaptation is designed to take 50 minutes total, with 5 minutes to get students set up in teams, 30 minutes for students to complete the problem set, and 15 minutes to discuss the answers together as a class.

Prerequisite Student Knowledge

Students should have an understanding of:

• SDS PAGE

• Radiolabeling

• Translation

• Translocation

• Signal sequences

• Necessity versus sufficiency

• Centrifugation

• Antibody use for protein detection

Prerequisite Teacher Knowledge

Instructors should have an in-depth understanding of:

• Research techniques

  • Homogenization and sonication

  • SDS PAGE

  • Radiolabeling

  • Photo-crosslinking

  • Immunoprecipitation

• Assays

  • How an ER import assay works

  • How to set up an experiment to determine sufficiency

  • Photo-crosslinking assay

• Scientific concepts

  • ER structure

  • Microsomes

  • Translation

  • Translocation

  • Signal sequences

  • Function of protease K

  • Function of Triton X

  • Necessity versus sufficiency

  • Crosslinking

We recommend using Alberts’s Molecular Biology of the Cell textbook, 7th ed. (7) to brush up on these topics if needed. Open access to an older edition of this textbook is provided by the National Center for Biotechnology Information (NCBI) Bookshelf.

Scientific Teaching Themes

Active Learning

The problem set follows the student-centered POGIL model, which is designed to help students work together to construct knowledge and understanding (6). In POGIL, not only do students discuss the material to gain knowledge, but they are required to use the knowledge and apply it to new scenarios. POGIL also positions the instructor as a facilitator because the instructor assists students in their problem solving rather than leading the problem solving (8).

Students engage in social metacognition, or the awareness and regulation of others’ thinking, while working through POGIL-style lessons (9). Examples of social metacognition include students correcting themselves or one another, asking questions to monitor their understanding, recognizing their points of confusion, requesting feedback on their ideas, and evaluating their answers. Social metacognition requires students to be engaged in discussions about the task or their understanding of the content. Social metacognition is also associated with higher-quality reasoning (9).

POGIL is designed for teams of three to four students; however, there is a possibility of free riding in teams of four (10). Free riding, or social loafing, is undesirable not only because it results in less learning for the free rider, it also negatively affects team dynamics (10). Studies have shown that the probability of social loafing decreases in smaller teams (10), so we recommend using teams of two if free riding is an issue in your classroom.

Class discussion also requires students to be actively engaged and ready to present answers. If no teams volunteer to present their answer, you can use a random number generator to call on a team to present. This method, sometimes known as cold calling, has been shown to increase student preparation and engagement in group discussions (11).

Assessment

Instructors measure learning informally by walking around the class while students are working. You can keep note of the concepts students find most challenging and focus on those during the class discussion. Instructors also measure learning by grading the problem sets after the class session. We find when students are graded for participation rather than accuracy, they discuss their ideas within their teams in a low stress environment and participate in peer scaffolding. When teams are graded for accuracy, they may spend less time explaining their ideas to each other in favor of spending time perfecting their answers to maximize points.

Students benefit from written feedback on their problem sets. It is up to you how much or little feedback you would like to write on the problem sets themselves—we find at minimum students appreciate when we mark what is correct, incorrect, and missing. This way students can follow up outside of class to ask further questions about their answers.

Instructors can lastly measure learning using a matched-pair question on a summative assessment (see Supporting File S1).

If you would like to measure student learning from this activity, we recommend providing isomorphic questions in an individual pre-test before implementing the activity, then administering matched-pair questions in an individual post-assessment, which could be included as part of an exam. Comparing the pre- and post-activity scores can provide evidence of student learning.

Students can assess their own learning by evaluating their answers. Evaluation is a metacognitive skill students can use while solving problems, and it involves determining whether their answer or work product is accurate, sufficient, and properly addresses the question or assignment. Most student teams use evaluation when working through POGIL-style problem sets (9); however, some teams may need extra reminders to evaluate their work. If student teams finish early, you can encourage them to look back at their problem set and evaluate whether they feel they addressed an entire question in their answer or whether they feel their answer is correct.

Students can also assess their learning by reviewing their graded problem set after it is returned to them. The corrections worksheet, which can be found on the last page of the problem set (Supporting File S2), is designed to ensure students review and understand their feedback.

Inclusive Teaching

POGIL involves assigning roles within teams, giving each student a specific job to do (8). We use the roles presenter, recorder, and manager (see descriptions the Class Session section in Lesson Plan). These roles promote inclusivity within teams because they ensure every student is invited to fully participate. The roles evenly distribute the logistical work that needs to be done (e.g., writing or presenting the answers), increase student buy-in, and help prevent any one student from dominating.

We recommend making randomized teams or choosing the teams yourself rather than allowing students to pick their own teams. This promotes inclusivity because these approaches increase the likelihood of exposure to more diverse points of view and more equal distribution of resources among teams (12). In addition, when classrooms freely divide into groups at PWIs (primarily white institutions), white students sometimes exclude Black students by choosing to work with other white students (13). Assigning or randomizing teams can prevent these instances of subtle racism, creating a more inclusive classroom (14).

Lesson Plan

See Table 1 for the teaching timeline.

Table 1. Teaching timeline for Protein Import Into the ER.

Instructor Prep

You will need to add images of the autoradiographs to the problem set. First, download the problem set (Supporting File S2) and the two source papers (links included in Supporting File S2). Use your computer’s software to take a screenshot of Figure 2 from the Dobberstein lab paper (3) and Figure 3a from the Rapoport lab paper (4). For the Dobberstein figure, crop the image so it excludes lanes 5 and 6 and the bottom row labeled pH 11. For the Rapoport figure, include the whole image. Insert them into the problem set in the indicated spots. Read the problem set and key to ensure you have a thorough knowledge of the answers. Be sure you have a strong understanding of the topics listed in the Prerequisite Teacher Knowledge section and spend time refreshing on these topics if needed.

Before class, color print enough of the problem sets (Supporting File S2) for every student to have a copy. Print out a key (Supporting File S3) for yourself and a blank copy (Supporting File S2) to use to present the answers at the end of the session.

Class Session

At the start of class, break students up into teams of three. We recommend either making randomized teams or choosing the teams yourself rather than instructing students to form their own teams. Randomized or instructor-created teams promote inclusivity in the classroom (see the Inclusive Teaching section in Scientific Teaching Themes). An effective way to form random teams is to take the total number of students, divide this number by three, and have students count off up to this number. If the total number of students is not divisible by three, make one or two teams of four, as POGIL is designed for teams of three to four students. There is a possibility that a team of four may result in free riding (10). If this occurs, we recommend making teams of two instead (see more information about free riding in the Active Learning section in Scientific Teaching Themes).

When the students move into their teams, assign them the roles of recorder, presenter, and manager. The recorder is responsible for writing down the team’s answers to turn in, the presenter is responsible for volunteering and presenting answers during the class discussion, and the manager is responsible for keeping the team on-task and ensuring every team member contributes to group discussion. In teams of four, an additional role should be assigned. We use “analyst” in this scenario; the analyst observes team dynamics and gives suggestions on how the team could better collaborate. You can find descriptions of these roles in Supporting File S2 of the CourseSource paper titled “Investigating the Function of a Transport Protein: Where is ABCB6 Located in Human Cells?” published in 2017 by Julie Stanton and Kathryn Dye (15).

An easy way to randomly assign roles is using birthdays—the first birthday in the calendar year is presenter, second birthday in the year is recorder, and last birthday is manager. Students will write their names in the slots on the problem set that correspond to their assigned roles.

After roles are assigned, direct the students to begin working through the problem set. After 5–10 minutes, start walking around the room to answer student questions or to check in on teams. We have found 5–10 minutes is an adequate amount of time for students to engage in interactive problem solving and to start pinpointing what they may need help with. When helping teams, instead of giving them the answer, ask questions that will point them towards pertinent information and guide their reasoning. Some examples of these questions are listed in the following section.

Problem Set

We used BioRender to create all the figures in the problem set.

Question 1

Question 1 introduces students to microsomes. This should act as a warmup question where students practice drawing on their prior knowledge to answer a critical question. Knowledge of homogenization and sonication is required to understand this question, so we direct students to use their electronic devices if they need a refresher on these research techniques. If students get stuck and do not know where to start, encourage them to try and recall what they know about various organelle structures and their membranes.

Question 2

Question 2 asks students to interpret an autoradiograph from an ER import assay. Question 2 can be challenging for students, as it requires them to combine their previous knowledge with brand new information. Students should be familiar with SDS-PAGE, radiolabeling, and signal sequences. Note that students familiar with western blots may confuse the autoradiograph for a western blot. It can be helpful to remind students about the difference in the way proteins are detected in each experimental method.

For question 2a, students will sometimes assume the only source of ribosomes in the entire assay is from the rough microsomes. These students likely overlooked the fact that the in vitro environment already had translation machinery (i.e., ribosomes) from the wheat germ extract. Teams who make this mistake usually identify the lack of ribosomes as the reason for the absence of mature protein in lane 1. If students provide this reasoning, encourage them to think about how the pre-protein was synthesized in lane 1, or advise them to reread the question. Additionally, for question 2a, some students will be curious about the smaller bands below Mu-CSF in lane 1. These are products from in vitro translation of truncated mRNA. Prior to in vitro translation, the researchers synthesized mRNA by in vitro transcription, which resulted in full length mRNA and truncated mRNA. If students ask about the smaller bands, praise them for their curiosity. Tell them that the researchers carried out in vitro transcription prior to in vitro translation, and that when we try to recreate cellular processes in vitro, we can anticipate that unexpected products might be made (e.g., truncated mRNA). Instruct students to focus on the larger bands for pre-sMU-CSF and Mu-CSF. Lastly, for question 2a, it is possible students will interpret the pre-sMU-CSF and Mu-CSF as experimental variables for testing whether the signal sequence is necessary for import into the microsome. If you find students making this interpretation, remind them that Mu-CSF is generated from pre-sMU-CSF and ask them to use their knowledge of ER import to explain how this occurs.

Question 2b can elicit a common misunderstanding: many students assume protease K (PK) targets specific proteins, such as the pre-protein. If students give this reasoning, start by telling them PK degrades all proteins. This will require them to reevaluate their answer and come up with another reasoning construct. If students continue to struggle with this question, ask them to share with you their answer to question 2a. If students did not make the conclusion in 2a that the mature protein is generated via signal sequence cleavage after import into microsomes, it will be very difficult for them to correctly solve question 2b. Start by giving the guidance listed above for question 2a. Once students correctly solve 2a, they will usually be able to follow the line of reasoning and determine the pre protein was degraded by PK in lane 3 because it was not protected by the microsome membrane. If they are having trouble reaching this conclusion, ask them to think about what type of macromolecule PK is (i.e., a protein) and whether proteins can diffuse across membranes.

The goal of question 2c is for students to understand how lane 4 operates as a control experiment. The most common misunderstanding for 2c is the assumption that Triton X (TX) can degrade proteins as well as membranes. If students offer this idea, remind them that TX only degrades membranes. For students who have the correct answers for 2a and 2b, this clarification should help them understand that PK is what degraded the mature protein after the protective membrane was degraded by TX. If students are still having trouble after you give this guidance, ask them to explain their answers for 2a and 2b, give them feedback on those, and ask them to adjust their reasoning for those before returning to 2c. Another common misunderstanding students may have is the assumption that the TX gets added at the same time as the microsomes. These students may incorrectly conclude there is no band in lane 4 because the microsomes were disrupted and were never able to generate the mature protein. If students offer this idea, gently ask them to reread the question again, as the question specifies that TX was added after the translation reaction. Not all teams will provide sufficient reasoning for why lane 4 is an important control. If students finish the problem set early, ask them to explain their answer to question 2c. If they do not address how lane 4 works as a control, encourage them to add this to their answer.

Question 3

While question 2 requires students to learn the details of the ER import assay, question 3 requires students to think about the overall purpose of the ER import assay and how to use it to design their own experiment. Students should be familiar with the difference between necessity and sufficiency; however, we have provided a slide they can use if they need a refresher (Supporting File S4; [16]). If students ask for more guidance after reading the slides, ask them questions about how they can take the provided definition of sufficiency and apply that to an ER signal sequence. Some examples could be:

What is the function of the signal sequence?

Students should answer that the signal sequence directs proteins to the ER.

What is an example of something that does not have that function?

The answer should be a protein that does not localize to the ER, such as a nuclear or mitochondrial protein.

How could you use a nuclear or mitochondrial protein to test the sufficiency of the signal sequence?

These questions should stimulate student thinking and lead them in the right direction.

For question 3, students will also need to come up with a way to test whether the protein localized to the ER. Some students may have the idea to use fluorescence microscopy and an ER marker protein. Congratulate them for having a good idea that would work in practice, and then ask them to instead use what they have learned thus far in the problem set to test for localization. This will encourage students to solidify and apply their knowledge of the ER import assay.

Question 4

Question 4 introduces two research techniques (i.e., crosslinking and immunoprecipitation) that may be new to students, and then it asks students to interpret data generated using these techniques. To understand the function of the experiment in question 4, students need to understand how co-immunoprecipitation works. We have found students respond well to a demonstration using a dry erase marker:

(Remove the cap from the marker.) Pretend this cap and this marker are two separate proteins, and we perform crosslinking to bind them (snap the cap onto the marker). If we use an antibody to immunoprecipitate the cap (hold the cap side of the marker and pull it down), both proteins will be pulled down. If we use an antibody to immunoprecipitate the marker (hold the marker side and pull down), both proteins will be pulled down.

You can decide whether to provide this demonstration to the whole class when teams start to answer question 4 or to individual groups if they ask about how the experiment works.

Question 4a can be challenging for students if they are unaware that the nascent protein gets released from the ribosome when the stop codon is reached. If a team is having trouble with 4a, ask them a question that will point them in the right direction, such as:

What happens when a stop codon reaches a ribosome?

In our experience, a team of three students is usually able to name that the nascent protein gets released from the ribosome. If students are still asking for assistance after this event is described, they may not have grasped that the experiment is aiming to crosslink the nascent protein with the translocon. Try asking them:

If the protein gets released, how does that affect crosslinking?

This question should help them make the connection between the aim of the experiment and the removal of the stop codon.

Question 4b requires students to apply their prior knowledge about SDS PAGE and autoradiography and their newly acquired knowledge about immunoprecipitation. Some students may say alpha F did not show up in lane 1 of the gel because it did not undergo irradiation. While this is technically true, students need to explain that the lack of irradiation made it so alpha F did not covalently bond with anything else, so due to its small size, it ran off the end of the gel (students should note the minimum protein size on this gel is 30 kDa). Other teams may assume that irradiation allows the proteins to be visible in the gel. In this case, ask students a question that points to the radioactive methionine, such as:

What is the difference between the radioactive methionine and the photo-lysine?

In question 4c, we have found virtually all teams correctly conclude alpha F interacts with Sec61 during translocation, suggesting Sec61 may form part of the translocon. Students may have a harder time explaining their reasoning if they have not fully grasped how crosslinking works. For their reasoning, students should explain that the bands in lanes 2 and 5 show proteins of the same size because in both lanes, alpha F has been crosslinked with Sec61, so the crosslinked protein complex can be immunoprecipitated with either anti-alpha F or anti-Sec61 antibodies. Students should also explain that because they crosslinked under UV irradiation, this means the two proteins were physically touching or in close proximity during irradiation.

Team Guidance

Some teams may finish the problem set before it is time for the class discussion. We have found it is helpful to approach early finishers and ask them about their reasoning for questions 2a, 2b, and 2c, because these questions involve many potential misunderstandings. This will also remind these teams of the value of evaluating their work.

Class Discussion

After about 50 minutes, most of the students will have finished the problem set, which leaves approximately 20 minutes to discuss answers. Start at the beginning of the problem set and ask if any team presenter would like to volunteer to share their answer. If possible, it would be helpful to use a document camera so students can show their answer to the class as they are verbally presenting it. After each presenter shares their answer, address their ideas, and explain the correct answer. Even if the presenter’s answer was spot on, it is helpful if you follow up and reword the answer, as the class will benefit from hearing differently worded explanations. If a student presents an idea that is incorrect or incomplete, ask if another team would like to volunteer to present their ideas. When addressing incorrect answers, we find it effective to highlight a correct idea within a student’s answer and elaborate using their idea as a starting point. This method provides clear instruction to the rest of the class while mitigating the risk of drawing negative attention to the presenter.

After the class session, ask the recorders from each team to turn in their problem set.

Problem Set Feedback

Students will need to sit with their teams when you return the problem set feedback. This will ensure every student can read the feedback and take photos of it for their own records. Instruct teams to send photos of the feedback to any absent team members via the email they provided on the problem set.

Give teams at minimum 10 minutes to work on the corrections on the last page of the problem set. This in-class time will give students the opportunity to ask you questions about their feedback. Depending on your class schedule, you could give teams even more time to work on their corrections or assign corrections for homework.

Teaching Discussion

Effectiveness in Achieving Learning Goals and Objectives

This lesson is an effective way for students to learn more about the experiments that helped the scientific community make important discoveries about protein import into the endoplasmic reticulum. In our experience, some student teams need guidance to generate the correct answers, particularly for questions 2a, 2b, 2c, and 4a. Some teams will use metacognition and evaluate their answers or determine they are confused, whereas others may not. This is why instructors should walk around the room and check in with teams to gauge the direction of their reasoning.

After completing this problem set, students perform well on exam questions related to the knowledge and skills they are meant to gain from the problem set. For example, during our most recent offering of the course, students achieved an average score of 86.8% on a challenging new crosslinking question designed to assess the knowledge and skills they gained from question 4. This question also required far transfer, meaning students had to integrate their knowledge and skills from question 4 with concepts covered in other parts of the class. In contrast, they would not have earned a high grade if we scored their group’s answers to the problem set for correctness prior to the whole-class discussion.

Formative assessment reveals that some students are still learning the specific meaning of photo-crosslinking experimental results; we have found some students state that a positive result indicates one protein is inside another. You can address this by explicitly stating that photo-crosslinking can reveal if two proteins are near one another, but it cannot provide evidence of the specific spatial arrangement of the two proteins.

Student and Instructor Reactions to the Lesson

This problem set can be challenging because it requires students to keep track of many experimental factors and apply new knowledge to interpret data. Still, the challenging nature of the problem set seems to increase student satisfaction when the reasoning clicks. Students show feelings of achievement and excitement when they take the “what” and piece it together with the “why” and “how,” for example why Triton X was used in the ER import assay and how that step functions as a control.

Instructors find this lesson facilitates not only content comprehension, but also skill development. We hear from instructors how this problem set helps students develop reasoning and experimental design skills. Instructors say the problem set highlights the importance of experimental controls and how chemical engineering techniques can be used to develop innovative biological experiments. We also hear how the lesson is engaging for students as the students remain on-task to complete the problem set.

Suggestions for Possible Adaptations

If your class period is less than 75 minutes, we suggest removing question 4 from the problem set or assigning question 4 as homework. Questions 1–3 provide a comprehensive lesson on the ER import assay, as question 2 asks students to explain each step of the assay and question 3 requires students to use the assay in an experiment they design themselves.

If you would like to extend the problem set for a class period longer than 75 minutes, we suggest adding the following questions:

2d. What would the results look like if the protease K the researchers used was expired (i.e., nonfunctional)?

3b. You note that a KG (Lys/Gly) dipeptide is found in the signal sequences of all the viruses’ secreted proteins. How could you determine if this KG dipeptide is required for cleavage of the signal sequence? What results would you get if the KG dipeptide is required for cleavage?

Supporting Materials

• S1. Protein Import into ER – Matched Pair Exam Questions

• S2. Protein Import into ER – Problem Set Student Version

• S3. Protein Import into ER – Problem Set Key [Instructor view only]

• S4. Protein Import into ER – Necessary and Sufficient Slides

Acknowledgments

We thank Dr. Vasant Muralidharan for his insights about the lesson. We thank Dr. Rachel Roberts-Galbraith for proofreading the manuscript.

References

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