Skip to main content

cell biology

Finding Beauty in Cell Stress

Posted on by

Most stressful situations that we experience in daily life aren’t ones that we’d choose to repeat. But the cellular stress response captured in this video is certainly worth repeating a few times, so you can track what happens when two cancer cells get hit with stressors.

In this movie of two highly stressed osteosarcoma cells, you first see the appearance of many droplet-like structures (green). This is followed by a second set of droplets (magenta) and, finally, the fusion of both types of droplets.

These droplets are composed of fluorescently labeled stress-response proteins, either G3BP or UBQLN2 (Ubiquilin-2). Each protein is undergoing a fascinating process, called phase separation, in which a non-membrane bound compartment of the cytoplasm emerges and constrains the motion of proteins within it. Subsequently, the proteins fuse with like proteins to form larger droplets, in much the same way that raindrops merge on a car’s windshield.

Julia Riley, an undergraduate student in the NIH-supported lab of Heidi Hehnly and lab of Carlos Castañeda, Syracuse University, NY, shot this movie using the sophisticated tools of fluorescence microscopy. It’s the next installment in our series featuring winners of the 2019 Green Fluorescent Protein Image and Video Contest, sponsored by the American Society for Cell Biology. The contest honors the discovery of green fluorescent protein (GFP), which—together with a rainbow of other fluorescent proteins—has enabled researchers to visualize proteins and their dynamic activities inside cells for the last 25 years.

Riley and colleagues suspect that, in this case, phase separation is a protective measure that allows proteins to wall themselves off from the rest of the cell during stressful conditions. In this way, the proteins can create new functional units within the cell. The researchers are working to learn much more about what this interesting behavior entails as a basic organizing principle in the cell and how it works.

Even more intriguing is that similar stress-responding proteins are commonly altered in people with the devastating neurologic condition known as amyotrophic lateral sclerosis (ALS). ALS is a group of rare neurological diseases that involve the progressive deterioration of neurons responsible for voluntary movements such as chewing, walking, and talking. There’s been the suggestion that these phase separation droplets may seed the formation of the larger protein aggregates that accumulate in the motor neurons of people with this debilitating and fatal condition.

Castañeda and Hehnly, working with J. Paul Taylor at St. Jude Children’s Research Hospital, Memphis, earlier reported that Ubiquilin-2 forms stress-induced droplets in multiple cell types [1]. More recently, they showed that mutations in Ubiquilin-2 have been linked to ALS changes in the way that the protein undergoes phase separation in a test tube [2].

While the proteins in this award-winning video aren’t mutant forms, Riley is now working on the sequel, featuring versions of the Ubiquilin-2 protein that you’d find in some people with ALS. She hopes to capture how those mutations might produce a different movie and what that might mean for understanding ALS.

References:

[1] Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions. Dao TP, Kolaitis R-M, Kim HJ, O’Donovan K, Martyniak B, Colicino E, Hehnly H, Taylor JP, Castañeda CA. Molecular Cell. 2018 Mar 15;69(6):965-978.e6.

[2] ALS-Linked Mutations Affect UBQLN2 Oligomerization and Phase Separation in a Position- and Amino Acid-Dependent Manner. Dao TP, Martyniak B, Canning AJ, Lei Y, Colicino EG, Cosgrove MS, Hehnly H, Castañeda CA. Structure. 2019 Jun 4;27(6):937-951.e5.

Links:

Amyotrophic Lateral Sclerosis (ALS) (National Institute of Neurological Disorders and Stroke/NIH)

Castañeda Lab (Syracuse University, NY)

Hehnly Lab (Syracuse University)

Green Fluorescent Protein Image and Video Contest (American Society for Cell Biology, Bethesda, MD)

2008 Nobel Prize in Chemistry (Nobel Foundation, Stockholm, Sweden)

NIH Support: National Institute of General Medical Sciences


The Perfect Cytoskeletal Storm

Posted on by

Ever thought about giving cell biology a whirl? If so, I suggest you sit down and take a look at this full-blown cytoskeletal “storm,” which provides a spectacular dynamic view of the choreography of life.

Before a cell divides, it undergoes a process called mitosis that copies its chromosomes and produces two identical nuclei. As part of this process, microtubules, which are structural proteins that help make up the cell’s cytoskeleton, reorganize the newly copied chromosomes into a dense, football-shaped spindle. The position of this mitotic spindle tells the cell where to divide, allowing each daughter cell to contain its own identical set of DNA.

To gain a more detailed view of microtubules in action, researchers designed an experimental system that utilizes an extract of cells from the African clawed frog (Xenopus laevis). As the video begins, a star-like array of microtubules (red) radiate outward in an apparent effort to prepare for cell division. In this configuration, the microtubules continually adjust their lengths with the help of the protein EB-1 (green) at their tips. As the microtubules grow and bump into the walls of a lab-generated, jelly-textured enclosure (dark outline), they buckle—and the whole array then whirls around the center.

Abdullah Bashar Sami, a Ph.D. student in the NIH-supported lab of Jesse “Jay” Gatlin, University of Wyoming, Laramie, shot this movie as a part his basic research to explore the still poorly understood physical forces generated by microtubules. The movie won first place in the 2019 Green Fluorescent Protein Image and Video Contest sponsored by the American Society for Cell Biology. The contest honors the 25th anniversary of the discovery of green fluorescent protein (GFP), which transformed cell biology and earned the 2008 Nobel Prize in Chemistry for three scientists who had been supported by NIH.

Like many movies, the setting was key to this video’s success. The video was shot inside a microfluidic chamber, designed in the Gatlin lab, to study the physics of microtubule assembly just before cells divide. The tiny chamber holds a liquid droplet filled with the cell extract.

When the liquid is exposed to an ultra-thin beam of light, it forms a jelly-textured wall, which traps the molecular contents inside [1]. Then, using time-lapse microscopy, the researchers watch the mechanical behavior of GFP-labeled microtubules [2] to see how they work to position the mitotic spindle. To do this, microtubules act like shapeshifters—scaling to adjust to differences in cell size and geometry.

The Gatlin lab is continuing to use their X. laevis system to ask fundamental questions about microtubule assembly. For many decades, both GFP and this amphibian model have provided cell biologists with important insights into the choreography of life, and, as this work shows, we can expect much more to come!

References:

[1] Microtubule growth rates are sensitive to global and local changes in microtubule plus-end density. Geisterfer ZM, Zhu D, Mitchison T, Oakey J, Gatlin JC. November 20, 2019.

[2] Tau-based fluorescent protein fusions to visualize microtubules. Mooney P, Sulerud T, Pelletier JF, Dilsaver MR, et al. Cytoskeleton (Hoboken). 2017 Jun;74(6):221-232.

Links:

Mitosis (National Human Genome Research Institute/NIH)

Gatlin Lab (University of Wyoming, Laramie)

Green Fluorescent Protein Image and Video Contest (American Society for Cell Biology, Bethesda, MD)

2008 Nobel Prize in Chemistry (Nobel Foundation, Stockholm, Sweden)

NIH Support: National Institute of General Medical Sciences


Seeing the Cytoskeleton in a Whole New Light

Posted on by

It’s been 25 years since researchers coaxed a bacterium to synthesize an unusual jellyfish protein that fluoresced bright green when irradiated with blue light. Within months, another group had also fused this small green fluorescent protein (GFP) to larger proteins to make their whereabouts inside the cell come to light—like never before.

To mark the anniversary of this Nobel Prize-winning work and show off the rainbow of color that is now being used to illuminate the inner workings of the cell, the American Society for Cell Biology (ASCB) recently held its Green Fluorescent Protein Image and Video Contest. Over the next few months, my blog will feature some of the most eye-catching entries—starting with this video that will remind those who grew up in the 1980s of those plasma balls that, when touched, light up with a simulated bolt of colorful lightning.

This video, which took third place in the ASCB contest, shows the cytoskeleton of a frequently studied human breast cancer cell line. The cytoskeleton is made from protein structures called microtubules, made visible by fluorescently tagging a protein called doublecortin (orange). Filaments of another protein called actin (purple) are seen here as the fine meshwork in the cell periphery.

The cytoskeleton plays an important role in giving cells shape and structure. But it also allows a cell to move and divide. Indeed, the motion in this video shows that the complex network of cytoskeletal components is constantly being organized and reorganized in ways that researchers are still working hard to understand.

Jeffrey van Haren, Erasmus University Medical Center, Rotterdam, the Netherlands, shot this video using the tools of fluorescence microscopy when he was a postdoctoral researcher in the NIH-funded lab of Torsten Wittman, University of California, San Francisco.

All good movies have unusual plot twists, and that’s truly the case here. Though the researchers are using a breast cancer cell line, their primary interest is in the doublecortin protein, which is normally found in association with microtubules in the developing brain. In fact, in people with mutations in the gene that encodes this protein, neurons fail to migrate properly during development. The resulting condition, called lissencephaly, leads to epilepsy, cognitive disability, and other neurological problems.

Cancer cells don’t usually express doublecortin. But, in some of their initial studies, the Wittman team thought it would be much easier to visualize and study doublecortin in the cancer cells. And so, the researchers tagged doublecortin with an orange fluorescent protein, engineered its expression in the breast cancer cells, and van Haren started taking pictures.

This movie and others helped lead to the intriguing discovery that doublecortin binds to microtubules in some places and not others [1]. It appears to do so based on the ability to recognize and bind to certain microtubule geometries. The researchers have since moved on to studies in cultured neurons.

This video is certainly a good example of the illuminating power of fluorescent proteins: enabling us to see cells and their cytoskeletons as incredibly dynamic, constantly moving entities. And, if you’d like to see much more where this came from, consider visiting van Haren’s Twitter gallery of microtubule videos here:

Reference:

[1] Doublecortin is excluded from growing microtubule ends and recognizes the GDP-microtubule lattice. Ettinger A, van Haren J, Ribeiro SA, Wittmann T. Curr Biol. 2016 Jun 20;26(12):1549-1555.

Links:

Lissencephaly Information Page (National Institute of Neurological Disorders and Stroke/NIH)

Wittman Lab (University of California, San Francisco)

Green Fluorescent Protein Image and Video Contest (American Society for Cell Biology, Bethesda, MD)

NIH Support: National Institute of General Medical Sciences


Defining Neurons in Technicolor

Posted on by

Brain Architecture
Credit: Allen Institute for Brain Science, Seattle

Can you identify a familiar pattern in this image’s square grid? Yes, it’s the outline of the periodic table! But instead of organizing chemical elements, this periodic table sorts 46 different types of neurons present in the visual cortex of a mouse brain.

Scientists, led by Hongkui Zeng at the Allen Institute for Brain Science, Seattle, constructed this periodic table by assigning colors to their neuronal discoveries based upon their main cell functions [1]. Cells in pinks, violets, reds, and oranges have inhibitory electrical activity, while those in greens and blues have excitatory electrical activity.

For any given cell, the darker colors indicate dendrites, which receive signals from other neurons. The lighter colors indicate axons, which transmit signals. Examples of electrical properties—the number and intensity of their “spikes”—appear along the edges of the table near the bottom.

To create this visually arresting image, Zeng’s NIH-supported team injected dye-containing probes into neurons. The probes are engineered to carry genes that make certain types of neurons glow bright colors under the microscope.

This allowed the researchers to examine a tiny slice of brain tissue and view each colored neuron’s shape, as well as measure its electrical response. They followed up with computational tools to combine these two characteristics and classify cell types based on their shape and electrical activity. Zeng’s team could then sort the cells into clusters using a computer algorithm to avoid potential human bias from visually interpreting the data.

Why compile such a detailed atlas of neuronal subtypes? Although scientists have been surveying cells since the invention of the microscope centuries ago, there is still no consensus on what a “cell type” is. Large, rich datasets like this atlas contain massive amounts of information to characterize individual cells well beyond their appearance under a microscope, helping to explain factors that make cells similar or dissimilar. Those differences may not be apparent to the naked eye.

Just last year, Allen Institute researchers conducted similar work by categorizing nearly 24,000 cells from the brain’s visual and motor cortex into different types based upon their gene activity [2]. The latest research lines up well with the cell subclasses and types categorized in the previous gene-activity work. As a result, the scientists have more evidence that each of the 46 cell types is actually distinct from the others and likely drives a particular function within the visual cortex.

Publicly available resources, like this database of cell types, fuel much more discovery. Scientists all over the world can look at this table (and soon, more atlases from other parts of the brain) to see where a cell type fits into a region of interest and how it might behave in a range of brain conditions.

References:

[1] Classification of electrophysiological and morphological neuron types in the mouse visual cortex. N Gouwens NW, et al. Neurosci. 2019 Jul;22(7):1182-1195.

[2] Shared and distinct transcriptomic cell types across neocortical areas. Tasic B, et al. Nature. 2018 Nov;563(7729):72-78.

Links:

Brain Basics: The Life and Death of a Neuron (National Institute of Neurological Disorders and Stroke/NIH)

Cell Types: Overview of the Data (Allen Brain Atlas/Allen Institute for Brain Science, Seattle)

Hongkui Zeng (Allen Institute)

NIH Support: National Institute of Mental Health; Eunice Kennedy Shriver National Institute of Child Health & Human Development


Caught on Video: Cancer Cells in Act of Cannibalism

Posted on by

Tumors rely on a variety of tricks to grow, spread, and resist our best attempts to destroy them. Now comes word of yet another of cancer’s surprising stunts: when chemotherapy treatment hits hard, some cancer cells survive by cannibalizing other cancer cells.

Researchers recently caught this ghoulish behavior on video. In what, during this Halloween season, might look a little bit like The Blob, you can see a down-for-the-count breast cancer cell (green), treated earlier with the chemotherapy drug doxorubicin, gobbling up a neighboring cancer cell (red). The surviving cell delivers its meal to internal compartments called lysosomes, which digest it in a last-ditch effort to get some nourishment and keep going despite what should have been a lethal dose of a cancer drug.

Crystal Tonnessen-Murray, a postdoctoral researcher in the lab of James Jackson, Tulane University School of Medicine, New Orleans, captured these dramatic interactions using time-lapse and confocal microscopy. When Tonnessen-Murray saw the action, she almost couldn’t believe her eyes. Tumor cells eating tumor cells wasn’t something that she’d learned about in school.

As the NIH-funded team described in the Journal of Cell Biology, these chemotherapy-treated breast cancer cells were not only cannibalizing their neighbors, they were doing it with remarkable frequency [1]. But why?

A possible explanation is that some cancer cells resist chemotherapy by going dormant and not dividing. The new study suggests that while in this dormant state, cannibalism is one way that tumor cells can keep going.

The study also found that these acts of cancer cell cannibalism depend on genetic programs closely resembling those of immune cells called macrophages. These scavenging cells perform their important protective roles by gobbling up invading bacteria, viruses, and other infectious microbes. Drug-resistant breast cancer cells have apparently co-opted similar programs in response to chemotherapy but, in this case, to eat their own neighbors.

Tonnessen-Murray’s team confirmed that cannibalizing cancer cells have a survival advantage. The findings suggest that treatments designed to block the cells’ cannibalistic tendencies might hold promise as a new way to treat otherwise hard-to-treat cancers. That’s a possibility the researchers are now exploring, although they report that stopping the cells from this dramatic survival act remains difficult.

Reference:

[1] Chemotherapy-induced senescent cancer cells engulf other cells to enhance their survival. Tonnessen-Murray CA, Frey WD, Rao SG, Shahbandi A, Ungerleider NA, Olayiwola JO, Murray LB, Vinson BT, Chrisey DB, Lord CJ, Jackson JG. J Cell Biol. 2019 Sep 17.

Links:

Breast Cancer (National Cancer Institute/NIH)

James Jackson (Tulane University School of Medicine, New Orleans)

NIH Support: National Institute of General Medical Sciences


Next Page