Misleading images in cell biologyJune 15, 2016
Light cannot be used to image any structures smaller than half its wavelength - for a long time, this was considered to be the ultimate resolution limit in light microscopy. The development of superresolution microscopy, however, showed that there are certain loopholes to this rule. By imaging individual molecules at different points in time, their exact positions can eventually be combined to one clear picture. In 2014, the Nobel Prize in Chemistry was awarded for this idea. Since then, superresolution microscopy techniques, such as STORM and PALM, have become popular methods to study the organization of proteins in the cell membrane.
Surprisingly, many research groups found that virtually all studied proteins form clusters in the cell membrane. During their work on such protein clusters, researchers at TU Wien made an unexpected discovery. Often, the alleged clusters are in fact single blinking molecules, which are counted multiple times. A new method, which the team has now presented in the journal Nature Methods, can distinguish real clusters from such artefacts.
Modern Imaging for Cell Biology
"For many important problems in medicine and biology, it is crucial to understand the structure of the cell membrane", says Florian Baumgart from the biophysics-research team led by Professor Gerhard Schütz at TU Wien. "Superresolution microscopy is an ideal tool to study the spatial arrangement of proteins on the cell membrane. Our research focuses on T cells, which can recognize antigens and therefore play an important role in our immune system."
In order to apply superresolution microscopy to biological samples, proteins are labeled with fluorescent molecules (so called fluorophores). In classical fluorescence microscopy, a single fluorophore is not visible as a clear dot, but as a washed-out circle. Consequently, if all these molecules light up simultaneously, their images overlap and the information about their exact spatial arrangement is lost. Using chemical tricks or special illumination protocols, the fluorophores can be made to light up at different time points. A series of pictures is taken, on each of which only a few well-separated fluorophores are visible. Subsequently, a computer program determines the exact position of the molecules on each image. This finally allows to reconstruct one high-resolution picture from all determined single molecule positions.
"In recent years, various research groups have investigated how proteins are distributed on cell membranes. Again and again they found that almost all proteins form clusters, rather than assuming random positions", says Florian Baumgart.
At first glance, this seems reasonable. It is well known that during antigen recognition, T cells can create stable protein clusters which are large enough to be seen even with classical fluorescence microscopy. Tiny nanoscopic protein clusters were considered to be precursors of these larger structures - with great importance for the function of T cells.
Florian Baumgart had been looking for these nanoclusters too. But superresolution microscopy is a complicated matter with many possible sources of error. The data must be evaluated carefully to obtain reliable results. Sometimes molecules can light up multiple times, which can easily be mistaken for a molecule cluster.
"We thought about how we could distinguish clusters of alternately glowing molecules from a single molecule which blinks repeatedly", says Florian Baumgart. The decisive idea was to vary the concentration of the fluorophores. "In the pictures, clusters and single blinking molecules may be difficult to tell apart, but the statistical properties of the distribution are different, when we increase the fluorophore concentration." If the labeled molecules do form clusters, more and more molecule positions become visible, when increasing amounts of fluorescent markers are used. But between the clusters, dark spaces remain. If, on the other hand, it is randomly distributed molecules lighting up several times, an increasing number of fluorescent molecules will eventually cover the whole surface, without forming clusters in any specific region.
"That way we found out that the membrane proteins we are interested in do not form clusters at all - this theory has to be discarded", says Florian Baumgart. "There are, however, examples of proteins that do indeed form clusters. We could show that pictures of these proteins have quite different statistical properties."
This method of distinguishing clusters from blinking molecules has now been published in the journal "Natures Methods". It is expected to help with the interpretation of superresolution data and might become an important tool to help in the understanding of the plasma membrane's structural organization.
Picture download: https://www.tuwien.ac.at/dle/pr/aktuelles/downloads/2016/truegerischepics/
Dr. Florian Baumgart
Institute for Applied Physics
Getreidemarkt 9, 1060 Vienna
Vienna University of Technology
Related Proteins Articles:
Scientists describe a well-defined mitochondrial proteome in baker's yeast.
Scientists from the University of Bristol have designed a new protein structure, and are using it to understand how protein structures are stabilized.
How did protein interactions arise and how have they developed?
Researchers recently confirmed it is possible to extract proteins from 80-million-year-old dinosaur bones.
Researchers at the Biozentrum of the University of Basel have developed a new method by which proteins can be transported to a new location in a cell.
Ancient proteins may offer clues on how to engineer proteins that can withstand the high temperatures required in industrial applications, according to new research published in the Proceedings of the National Academy of Sciences.
Florian Praetorius and Professor Hendrik Dietz of the Technical University of Munich have developed a new method that can be used to construct custom hybrid structures using DNA and proteins.
EPFL scientists have carried out a genomic and evolutionary study of a large and enigmatic family of human proteins, to demonstrate that it is responsible for harnessing the millions of transposable elements in the human genome.
Some organisms are able to survive in hot springs, while others can only live at mild temperatures because their proteins aren't able to withstand such extreme heat.
Small 'bubbles' frequently form on membranes of cells and are taken up into their interior.
Related Proteins Reading:
Proteins: Structure and Function
by David Whitford (Author)
Proteins: Structure and Function is a comprehensive introduction to the study of proteins and their importance to modern biochemistry. Each chapter addresses the structure and function of proteins with a definitive theme designed to enhance student understanding. Opening with a brief historical overview of the subject the book moves on to discuss the ‘building blocks’ of proteins and their respective chemical and physical properties. Later chapters explore experimental and computational methods of comparing proteins, methods of protein purification and protein folding and... View Details
Clean Protein: The Revolution that Will Reshape Your Body, Boost Your Energy—and Save Our Planet
by Kathy Freston (Author), Bruce Friedrich (Author)
Join the CLEAN PROTEIN revolution and lose weight, feel stronger, and live longer.
Food and wellness experts Kathy Freston and Bruce Friedrich have spent years researching the future of protein. They've talked to the food pioneers and the nutrition scientists, and now they've distilled what they've learned into a strength-building plan poised to reshape your body and change your world.
Complete with delicious recipes and a detailed guide to food planning, Clean Protein explains everything you need to know in order to get lean, gain energy, and stay mentally sharp.... View Details
Protein Power: The High-Protein/Low Carbohydrate Way to Lose Weight, Feel Fit, and Boost Your Health-in Just Weeks!
by Michael R. Eades (Author), Mary Dan Eades (Author)
New York Times Bestseller - An effective, medically sound diet that lets you eat bacon, eggs, steak, even cheese? It's true! Lose fat. Feel fit. Stop craving. Without counting fat grams and without giving up the foods you love. Includes recipes for healthy meals to lose weight.
Based on cutting-edge research, this revolutionary and deliciously satisfying plan has already helped thousands of patients lose weight and achieve other lifesaving health benefits, including lower cholesterol and blood pressure readings and an improvement or reversal of common... View Details
Proteins: Concepts in Biochemistry
by Paulo Almeida (Author)
Proteins: Concepts in Biochemistry teaches the biochemical concepts underlying protein structure, evolution, stability, folding, and enzyme kinetics, and explains how interactions in macromolecular structures determine protein function. Intended for a one-semester course in biochemistry or biophysical chemistry with a focus on proteins, this textbook emphasizes the logic underlying biophysical chemical principles.
Problems throughout the book encourage statistical and quantitative thinking. The text is ideal for senior undergraduates, first year graduate students, and... View Details
The Protein Power Lifeplan
by Michael R. Eades (Author), Mary Dan Eades (Author)
Introduces a lifestyle program that includes motivational advice, recipes, health tips, and nutritional guidelines to assist in treating major health problems, including diabetes, obesity, hypertension, and heart disease View Details
Protein (Healthy Eating with MyPlate)
by Nancy Dickmann (Author)
Make a place for protein in your diet. Readers will learn that leaner is better when it comes to choosing the healthiest meat or protein for their meals. Read this book to learn about how to eat well and use MyPlate. View Details
Vegetarian Times Plant-Powered Protein Cookbook: Over 200 Healthy & Delicious Whole-Food Dishes
by Editors of Vegetarian Times (Author)
Healthy, Delicious Recipes with Plant Powered Protein
Protein is the macronutrient that’s on the tip of everyone’s tongue these days, but a protein-rich diet doesn’t have to mean chowing down on giant bowls of beans or plates of egg whites—nor does it have to include meat, fish, or poultry. In this book, the Vegetarian Times editors set the protein story straight, showing you how to meet all your protein needs with delicious, satisfying, easy-to-prepare recipes that fit seamlessly into any lifestyle.... View Details
The High-Protein Vegetarian Cookbook: Hearty Dishes that Even Carnivores Will Love
by Katie Parker (Author), Kristen Smith (Author)
Satisfying vegetarian recipes from Veggie and the BeastWhere do vegetarians get their protein? From delicious plant-based foods, including beans, nuts, quinoa, raw cocoa, and even dairy. These ingredients are used to their best advantage in this new cookbook. As a vegetarian living with a meat-eating guy, the author has developed recipes for every time of day (or night) that are deliciously satisfying and high in protein. With recipes like Fresh Veggie Quinoa Salad with Lemon Tahini Dressing, Mushroom and Wild Rice Burgers, Quick and Hearty Vegetarian Chili,... View Details
The Oil-Protein Diet Cookbook
by Johanna Budwig (Author)
A healthy food preparation guide by a seven-time Nobel Prize nominee and Europe's foremost authority on cancer and nutrition.
Contemporary nutritional science owes a great deal to Dr. Budwig's early discoveries on fat metabolism and healing.
This brilliant scientific mind has put together a wonderfully imaginative best seller "cookbook" --a guide for the use of healthy oils in daily meal preparation.
Not only will readers discover over 500 delicious meal possibilities using the healing powers of flax oil, they will also learn more about how "good" fats and "bad" fats... View Details
The Protein Counter 3rd Edition
by Jo-Ann Heslin M.A. R.D. CDN (Author), Karen J Nolan Ph.D. (Author)
MORE THAN 7.5 MILION COUNTER BOOKS IN PRINT FROM THE NUTRITION EXPERTS
Put the latest protein recommendations to work for you.
Every day your body must build and replace millions of cells—an impossible job without proteins. The amount you need changes with exercise, stress, weight loss, illness, injury, and pregnancy. That’s why your body is counting on you to eat the proteins it needs to maximize fitness, boost your immune system, protect you from chronic disease, help you lose weight and keep it off, and much more. The completely revised and updated Protein... View Details