First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Rasha Khaddaj is first author on ‘ The surface of lipid droplets constitutes a barrier for endoplasmic reticulum-resident integral membrane proteins’, published in JCS. Rasha is a postdoc in the lab of Roger Schneiter at the Department of Biology, University of Fribourg, Switzerland, investigating lipid droplet biogenesis in budding yeast.

Rasha Khaddaj

How would you explain the main findings of your paper in lay terms?

I am using yeast, the same type used to bake bread, to study how and where lipid droplets (LDs) are formed. LDs are found in both yeast and humans, and are a compartment dedicated to storing excess fat. They are unique organelles surrounded by a monolayer of phospholipids. In humans, they are linked to lipid-related diseases, such as cardiovascular disease, type 2 diabetes and obesity. It is therefore important to study this organelle in more detail.

In this study, we tested whether endoplasmic reticulum (ER)-resident integral membrane proteins can move from the lipid bilayer membrane of the ER onto the lipid monolayer of LDs. For this, we used fusion proteins between ER-resident proteins, such as Wbp1 or Sec61, and perilipin 3 (PLIN3), a mammalian LD protein. Interestingly, our fusion proteins localize to the LD periphery in both yeast and mammalian cells, as shown by confocal microscopy. However, this LD peripheral localization is not due to relocation of the reporter proteins onto the LD surface but rather is triggered by rearrangement of the ER membrane. We conclude that the surface membrane of LDs constitutes a barrier for bilayer transmembrane proteins.

Were there any specific challenges associated with this project? If so, how did you overcome them?

The main challenge we had to address in this work was whether the LD perimeter is continuous with the ER membrane. Despite being informative, fluorescence microscopy cannot resolve the space between the ER membrane and the LD monolayer due to limitations in resolution. We thus used two approaches to overcome this challenge. First, we fused Pmt1, an ER-resident integral membrane protein that has its C terminus inside the lumen of the ER, to PLIN3 and checked by microscopy whether the resulting fusion protein localizes to the droplet surface. We observed that the fused Pmt1 is homogeneously distributed in the ER and does not localize to the LD perimeter. Second, we used bimolecular fluorescence complementation (BiFC) between proteins localized to the ER and the LD surface. This approach allows us to visualize the spatial interaction of the proteins. These observations thus led to the conclusions that LDs constitute a barrier for bilayer-spanning proteins and that the ER membrane is wrapping the LD surface.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

I was astounded the first time I visualized how the crescent structures formed at the ER–LD interface changed to a full circle around the LD rim upon expression of membrane-anchored PLIN3. It was the moment where we resolved that our system triggered rearrangement of the ER membrane around the LD.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

I have had exceptional mentors during my research career. I was inspired by my first mentor Professor Julnar Usta in the Department of Biochemistry at the American University of Beirut, and by seeing how organized and knowledgeable she is. Following this, my PhD supervisor, Professor Roger Schneiter in the Department of Biology at the University of Fribourg, has not only taught me science but also helped me to grow as a person.

Expression of membrane-anchored PLIN3 extends the surface of interactions between Wbp1–VN and Erg6–VC to the entire LD perimeter. Cells expressing the indicated split-Venus fusions together with or without the membrane-anchored PLIN3, Wbp1–mScarlet–PLIN3, were cultivated in oleic acid-containing medium and analyzed by fluorescence microscopy. Yellow boxes indicate regions shown at higher magnification in the panels below. Note the crescent-shaped signal (orange arrowhead) from BiFC partially overlaps with the circular localization of Erg6–mCherry (white arrowhead), whereas the circular signal from BiFC (blue arrow) overlaps with the circular localization of the mScarlet-tagged membrane-anchored PLIN3. Scale bars: 2.5 µm. Illustration (bottom) of the crescent-shaped and circular-shaped BiFC signals representing extensive contacts between the ER membrane, where Wbp1–VN is localized, and LDs harboring Erg6–VC.

Expression of membrane-anchored PLIN3 extends the surface of interactions between Wbp1–VN and Erg6–VC to the entire LD perimeter. Cells expressing the indicated split-Venus fusions together with or without the membrane-anchored PLIN3, Wbp1–mScarlet–PLIN3, were cultivated in oleic acid-containing medium and analyzed by fluorescence microscopy. Yellow boxes indicate regions shown at higher magnification in the panels below. Note the crescent-shaped signal (orange arrowhead) from BiFC partially overlaps with the circular localization of Erg6–mCherry (white arrowhead), whereas the circular signal from BiFC (blue arrow) overlaps with the circular localization of the mScarlet-tagged membrane-anchored PLIN3. Scale bars: 2.5 µm. Illustration (bottom) of the crescent-shaped and circular-shaped BiFC signals representing extensive contacts between the ER membrane, where Wbp1–VN is localized, and LDs harboring Erg6–VC.

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What's next for you?

As cardiovascular disease and obesity are the most prominent diseases in the world, I would like to continue research in the lipid field. This allows me to contribute to paving the way to development of new treatments, which could help people suffering from such diseases.

Tell us something interesting about yourself that wouldn't be on your CV

I like to explore new cultures and meditation. The latter not only makes you joyful but also creates a space between you and what is your mind and body, which helps with being focused and disciplined in research.

What is the most important piece of advice you would give to first-year PhD students?

If I had to give one piece of advice to a first-year PhD student, it would be to read as much as they can and to set measurable, realistic goals. Science is mesmerizing, and most of the time when answering a scientific question, we enter into a turmoil of unending ideas that could be either helpful or misleading. For that, a clear mind accompanied by knowledge can help to more quickly find the answers to important questions.

What changes do you think could improve the professional lives of early-career scientists?

Competition is usually good motivation to work harder and learn more; however, it can be counterproductive. For that reason, I believe that collaboration between scientists and sharing knowledge, ideas and information help to improve the professional life of early-career scientists.

Rasha Khaddaj's contact details: Department of Biology, University of Fribourg, Chemin du Musée 10, 1700 Fribourg, Switzerland.

E-mail: [email protected]

Khaddaj
,
R.
,
Mari
,
M.
,
Cottier
,
S.
,
Reggiori
,
F.
and
Schneiter
,
R.
(
2022
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The surface of lipid droplets constitutes a barrier for endoplasmic reticulum-resident integral membrane proteins
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J. Cell Sci.
135
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jcs256206
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