November 26, 2014

 

Patterns are soothing for left-brained folks like me, with the exception being those terrible patterned holiday sweaters that will come out of mothball-ridden closets soon (unsettling for everyone, really). Today’s images are from a paper describing a new micropatterning technique to look at plasma membrane proteins. 

The plasma membrane of a cell is riddled with many multi-protein complexes that facilitate communication and transport. These complexes provide a challenge to biologists due to their ubiquitous localization around the cell, their large, complex size, and their transient interactions with proteins. A recent paper describes a technique to study signaling complexes at the plasma membrane, using micropatterns within the plasma membrane. Löchte and colleagues expressed a protein bait in a micropattern in a living cell’s plasma membrane. Dynamics of the interactions between the protein bait and ligand target can then be quantified using live microscopy and on a single-molecule level. Löchte and colleagues used the IFN interferon signaling complex, using one of the receptor units (IFNAR2) as bait. In the top images above, the micropatterned receptor bait (IFNAR2) was able to recruit the other IFN receptor subunit (IFNAR1, green) after the addition of the ligand (red; time represents the addition of the ligand). Bottom image shows a closer view of the high-affinity, micropatterned binding that requires simultaneous interaction of the ligand (red) with both receptor subunits.


Lochte, S., Waichman, S., Beutel, O., You, C., & Piehler, J. (2014). Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane originally published in the Journal of Cell Biology, 207 (3), 407-418 DOI: 10.1083/jcb.201406032

March 19, 2013

After a lovely Spring Break with my family, HighMag is back in action.  As an oldie, Spring Break takes on a whole new meaning than it did years ago…this pregnant lady didn’t have one single beer!

 Cancer is a series of cellular mistakes, mistakes that are too far gone to fix without the help of the medical field.  Understanding the mistakes at the most basic cellular level is key to fighting the war on cancer, and a recent paper is a fine example of this.

A great model for understanding cell polarity is the intestinal epithelial sheet of cells that provides a barrier between the inside of the intestine and the body.  The different polarized domains – apical and basal – each have a discrete set of adhesion and membrane proteins trafficked to them.  One membrane trafficking protein, Rab25, is a tumor suppressor for colon cancer in both humans and mice.  A recent paper investigates the link between Rab25 and the polarized intestinal cells involved in colon cancer.  Krishnan and colleagues found that a reduction of Rab25 levels in cultured endothelial cells resulted in increased cell invasion and a loss of certain integrins, adhesion proteins, at the plasma membrane.  Rab25 loss also affected the transcription of several genes including the transcription factor ETV4, suggesting that Rab25’s effect on cell polarity is through gene regulation.  The scanning electron images above show brush border microvilli, the fingerlike-projections seen in intestinal epithelial sheets that serve to increase absorption of nutrients from the intestine.  In Rab25-reduced cells (middle row), the brush border is sparsely-packed and disorganized when compared to control cells (top row).  Bottom row shows Rab25-reduced cells in which rabbit Rab25 was reintroduced, and the rescued brush border is more organized.

Krishnan M, Lapierre LA, Knowles BC, & Goldenring JR (2013). Rab25 regulates integrin expression in polarized colonic epithelial cells. Molecular biology of the cell, 24 (6), 818-31 PMID: 23345591

January 17, 2013

Membranes have to wear many hats. A cell’s plasma membrane must be sturdy enough to protect the cell, yet fluid enough to support the cell’s dynamic and swingin’ lifestyle. A recent paper hypothesizes how plasma membranes can accomplish both tasks, and provides beautiful images and results to show this fascinating life of a membrane.
 
A cell’s plasma membrane protects and contains the contents of the cell, but is also flexible and fluid enough to allow the many events that take place at or involve the membrane, such as cell migration and changes in cell shape. A recent paper presents a mechanism for how a plasma membrane can accomplish both structure and flexibility. Kapustina and colleagues monitored rounded cells and the periodic membrane protrusions they make, and found compression (folding) and dilation (unfolding) of the plasma membrane and underlying actin cortex during protrusion events. This compression-dilation mechanism allows the cell to make rapid changes in cell shape, and can produce amoeboid-like migration movements under certain conditions. The electron microscopy images above show a cell fixed during membrane oscillations. Membrane folding appears as finger-like or round projections. (The yellow box shows the position of the higher magnification image on the right; red arrows point to dense cortical regions.)

BONUS!!  Beautiful movie of F-actin (green) and myosin (red) during membrane protrusions is below.  More movies of these membrane dynamics can be seen here.

 Kapustina, M., Elston, T., & Jacobson, K. (2013). Compression and dilation of the membrane-cortex layer generates rapid changes in cell shape originally published in the Journal of Cell Biology, 200 (1), 95-108 DOI: 10.1083/jcb.201204157

November 6, 2012

It is Election Day here in the US, which means that some of us need stress relief until the results are tallied. So, maybe you can imagine the graceful membrane dynamics of a cell and let their lava-lamp-like groove lull you into a happy place. Today’s image is from a paper describing the regulation of caveola biogenesis.

Caveolae are small invaginations on a cell’s plasma membrane that play important roles in cell signaling and endocytosis (the uptake of material into a cell). Caveolae depend on proteins called caveolins, but the details of caveola biogenesis are not completely understood. A recent paper describes results showing the importance of phosphorylation of Caveolin-1 (Cav1) in the formation of caveolae. Phosphorylation is the addition of a phosphate group to a protein, which functions as a molecular switch to change the protein’s activity. Joshi and colleagues found that the phosphorylation of Cav1 induces a feedback loop that links together mechanical stress on the cell, caveola biogenesis, and focal adhesion regulation at the cell’s membrane. These results place Cav1 on a list of critical proteins that help a cell respond to mechanical stress. In the images above, mutations of Cav1 that mimic phosphorylation (Cav1Y14R and Cav1Y14D) cause the formation of more caveolae and caveolae clusters (arrows and asterisks) than control cells (Cav1WT).

Joshi, B., Bastiani, M., Strugnell, S., Boscher, C., Parton, R., & Nabi, I. (2012). Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation originally published in the Journal of Cell Biology, 199 (3), 425-435 DOI: 10.1083/jcb.201207089

October 11, 2012

When in a crowded elevator, mall, or football stadium, I panic knowing that I’d be the first person trampled and/or eaten in an emergency. Crowded places give me the willies, but when proteins are over-crowded, interesting things happen. Today’s image is from a paper showing what can happen at the membrane when proteins are over-crowded. 

Membrane bending is an essential part of many cellular events, including endocytosis and filopodia formation. It has been suggested that membrane curvature can stem from two mechanisms—the use of curved proteins to form a curved scaffold for the membrane, or the insertion of wedge-shaped hydrophobic helices into the membrane. A recent paper shows a third mechanism that can drive membrane curvature. Stachowiak and colleagues found that protein-protein crowding drives membrane bending. Protein coverage at 20% is sufficient to drive curvature, by creating lateral pressure of membrane-bound proteins colliding. Interestingly, even proteins unrelated to membrane curvature can induce curvature when overcrowded (GFP, for example). The cartoons and images above show vesicles that contain low (left) or high (right) levels of protein binding at specific domains at the membrane. High levels of protein caused membrane curvature, as seen as long lipid tubules.

Stachowiak JC, Schmid EM, Ryan CJ, Ann HS, Sasaki DY, Sherman MB, Geissler PL, Fletcher DA, & Hayden CC (2012). Membrane bending by protein-protein crowding. Nature cell biology, 14 (9), 944-9 PMID: 22902598 
Adapted by permission from Macmillan Publishers Ltd, copyright ©2012

September 20, 2012

Once an egg finds its dance partner, the incredible changes that take place have fascinated biologists for centuries. These changes are kicked off immediately, and thanks to modern microscopy we get to see them up close. Today’s image is from a paper describing actin-mediated changes at the membrane of an egg shortly after fertilization.

Shortly after fertilization, actin assembly and dynamics drive changes in membrane physiology in the egg. One of these changes is an increase in efflux from the egg, or transport out of the egg, at the same time that short actin-based protrusions called microvilli are assembled around the egg. A recent paper investigates this link between efflux and microvilli after fertilization of sea urchin eggs. Whalen and colleagues found that a protein called ABCB1a translocates to the tips of microvilli shortly after fertilization. ABCB1a is an ABC transporter, a family transmembrane proteins that drive movement of various material into and out of many cell types. By using structured illumination microscopy (3D-SIM), Whalen and colleagues captured high-resolution images of actin filaments shortly after fertilization, and found movement of ABCB1a along growing actin filaments. Both efflux activity and movement of ABCB1a to microvilli tips were inhibited after actin polymerization was blocked. The images above show actin filaments (green) and ABCB1a (red) at different time points shortly after fertilization. By 60 minutes after fertilization, ABCB1a moved from below microvilli to the tips of the protrusions. 

Kristen Whalen, Adam M. Reitzel, & Amro Hamdoun (2012). Actin polymerization controls the activation of multidrug efflux at fertilization by translocation and fine-scale positioning of ABCB1 on microvilli Molecular Biology of the Cell, 23 (18), 3663-3672 DOI: 10.1091/mbc.E12-06-0438

September 4, 2012

Membranes are not just cute little jackets that cells wear to keep them protected. Membranes are no joke—they are dynamic hotspots of action, and their composition plays a key role in many cellular processes. Today’s image is from a paper describing the composition of a cell’s plasma membrane during cell morphogenesis.

The cell’s plasma membrane serves as the entryway and exit for material and plays an important role in cell signaling. Although all plasma membranes are made of a bilayer of lipids, those lipids can vary depending on the type of cell. The local composition of lipids in the plasma membrane can affect cellular processes, as seen in the human pathogenic fungus human C. albicans. C. albicans can grow using a filamentous protrusion called a hypha, which was recently found to have an asymmetric distribution of lipids. According to Vernay and colleagues, the growth of the hyphal filament was accompanied by a gradient of the phospholipid PI(4,5)P2 (phosphoinositide bis-phosphate). PI(4,5)P2 is frequently found within membranes at the sites of polarized growth, trafficking, and actin reorganization. Vernay and colleagues found that the sharp gradient of PI(4,5)P2 is essential for proper cell morphogenesis of C. albicans, and depends on the actin cytoskeleton and PI(4)P synthesis. In the images above, hyphal filaments show a gradient of PI(4,5)P2 using a color-coding of PI(4,5)P2 signal (key shows relative levels). Highest PI(4,5)P2 levels are seen at the tips of the filaments.

Aurélia Vernay, Sébastien Schaub, Isabelle Guillas, Martine Bassilana, & Robert A. Arkowitz (2012). A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth originally published in the Journal of Cell Biology, 198 (4), 711-730 DOI: 10.1083/jcb.201203099

August 30, 2012

The time and energy required to understand any complex process in the cell can be intimidating. Frequently biologists will take a systematic approach, whether that is identifying every protein involved, finding the exact location of proteins, or uncovering every protein-protein interaction, etc. Some biologists combine a few approaches, throw in their own clever spin, and turn out a pretty darn complete picture of what’s going on. With that in mind, enjoy today’s image!

Endocytosis is the uptake of material into the cell, and involves a long list of proteins that are recruited to and dissociated from the membrane in a specific order. A recent paper describes how the combination of fluorescent microscopy and electron microscopy was used to systematically correlate the composition of proteins with the plasma membrane’s shape throughout endocytosis in budding yeast. Kukulski and colleagues used fluorescent tags that correlate with different modules, or stages, of endocytosis to understand how the localizations of proteins overlap over time. Then, these fluorescent tags were used as a guide in electron tomography to visualize the exact structure and shape of the membrane at each module. In the images above, yeast cells have fluorescent tags for the coat module, an intermediate step, of endocytosis (green, Sla1) and a later actin module (red, Abp1). When the two proteins overlap at the membrane, the signal appears yellow. Below each fluorescent image is the corresponding electron tomography image that shows the ultrastructure of the membrane at each stage. The cartoon above shows the time window in endocytosis for each protein.

Wanda Kukulski, Martin Schorb, Marko Kaksonen, & John A.G. Briggs (2012). Plasma Membrane Reshaping during Endocytosis Is Revealed by Time-Resolved Electron Tomography Cell, 150 (3), 508-520 DOI: 10.1016/j.cell.2012.05.046

Copyright ©2012 Elsevier Ltd. All rights reserved.

June 21, 2012

There’s a song on the radio that you just love right now. You’re not sure what it is about the song that you love…the catchy chorus, the singer’s silky voice, the booming bass line, the touch of pan flute, whatever. If you’re musically-inclined, you start teasing the song apart in your head until you finally realize what it is (always, it’s the pan flute!). Knowing how to tease a complex problem apart is a key skill for any scientist, and today’s image is from a paper that pares down clathrin-coated pits to their bare minimum.

Clathrin coats are assembled cages of the scaffolding protein clathrin. These scaffolds deform a planar membrane into a curved membrane that is able to bud off during the uptake of material into the cell, a process called endocytosis. It was previously thought that clathrin-associated proteins helped to induce curvature in the membrane, but recently a group finds otherwise, in a study that describes the minimum requirements for clathrin-coated bud formation. In this paper, Dannhauser and Ungewickell monitored bud and vesicle formation using a cell-free system composed of brain lipids that artificially form vesicles called liposomes. They found that clathrin alone is sufficient to generate buds in a lipid membrane. In the images above, buds and vesicles were formed when liposomes were in the presence of clathrin, but lacking any other proteins able to induce membrane curvature. Many vesicles were, in fact, still attached to the liposomes via narrow membrane necks (arrows). 

Philip N. Dannhauser, & Ernst J. Ungewickell (2012). Reconstitution of clathrin-coated bud and vesicle formation with minimal components Nature Cell Biology, 14 (6), 634-639 : 10.1038/ncb2478

Adapted by permission from Macmillan Publishers Ltd, copyright ©2012

April 9, 2012

Membranes really know how to mingle. They are dynamic in the cell—budding away from one organelle to fuse with another, remodeling themselves for the situation. My awkward party persona should take some notes…I’ve never seen membranes hang out next to the Chex Mix bowl all night. Today’s image is from a recent paper on membrane scission and the role of membrane remodeling proteins.

The membranes that compartmentalize a cell’s organelles are under constant transformation. Membrane sculpting is a coordinated process that includes bending membranes and remodeling by fission and fusion (severing and joining, respectively). Membrane curvature is introduced two different ways—by hydrophobic insertions into the membrane’s lipid bilayer, or by the presence of a scaffold made of curved BAR domain proteins. A recent paper sheds light on how these two membrane-remodeling mechanisms affects membrane fission. According to Boucrot and colleagues, the membrane insertion of a protein called epsin, which contains a hydrophobic ENTH domain, leads to membrane fission, while the curved BAR-domain scaffolds actually limit membrane fission. In addition, epsin is required for membrane fission of clathrin-coated vesicles. The electron micrograph above shows a clathrin-coated vesicle after depletion of all epsin proteins. Without epsin, vesicles are unable to bud from one another, creating a multi-headed structure.

Boucrot, E., Pick, A., Çamdere, G., Liska, N., Evergren, E., McMahon, H., & Kozlov, M. (2012). Membrane Fission Is Promoted by Insertion of Amphipathic Helices and Is Restricted by Crescent BAR Domains Cell, 149 (1), 124-136 DOI: 10.1016/j.cell.2012.01.047
Copyright ©2012 Elsevier Ltd. All rights reserved.

February 2, 2012

When a cell makes it all the way to cytokinesis, it has already achieved greatness. DNA replication and mitosis are Big Deals, but a cell exits mitosis only to find itself in front of that final all-uphill mile of the New York City Marathon (even as a kid watching it, I thought that was so cruel). There is a lot of regulation and reorganizing that happens for a cell to correctly complete cell division and physically split into two cells, and a recent paper sorts out how membrane trafficking proteins are coordinated during the process.

Cytokinesis is the final step of cell division, when the two daughter cells are physically divided. At the start of cytokinesis, a contractile ring forms around the center of the dividing cell and begins to tighten. These contractions result in a cleavage furrow forming and pinching the dividing cell, after which only an intercellular bridge connects the two new daughter cells. With all of this contracting and pinching of the plasma membrane, it is no surprise that membrane trafficking proteins are important during cytokinesis. A recent paper looks at how endocytosis and membrane trafficking pathways are coordinated during cytokinesis. Specifically, Chesneau and colleagues found that Rab35 GTPase, an endocytic protein known to also be important in cytokinesis, is negatively regulated by ARF GTPase. ARF mutants (ones that are stuck in the activated GTP state, for those paying attention) have cytokinesis defects similar to Rab35 mutants (stuck in the inactive GDP state), including a failure after cleavage furrow progression and an instability of intercellular bridges. A seen in the images above, both the ARF mutant (bottom) and Rab35 mutant (middle) mislocalize a protein called SEPTIN2 (green, arrowheads), which is a cytoskeletal element that provides structure during cytokinesis. In a normal cell (top), SEPTIN2 is localized at the cleavage furrow.

Chesneau, L., Dambournet, D., Machicoane, M., Kouranti, I., Fukuda, M., Goud, B., & Echard, A. (2012). An ARF6/Rab35 GTPase Cascade for Endocytic Recycling and Successful Cytokinesis Current Biology DOI: 10.1016/j.cub.2011.11.058

Copyright ©2011 Elsevier Ltd. All rights reserved.

January 30, 2012

As the flu pins you to your bed this winter, take a feverish minute to thank the biologists who help us understand the virus that causes it, the influenza virus. And maybe make a promise to yourself that next year you’ll spend 10 minutes to get the vaccine.

Influenza is an RNA virus that causes fever, chills, pain, fatigue, and general misery. After the virus replicates inside a host cell, it assembles at the cell’s plasma membrane. Virus particles then bud from the cell’s plasma membrane, taking some of the membrane with it, and search for the next cell to invade. A recent paper describes the membrane composition of recently produced influenza virus particles, and suggests lipid raft involvement in influenza virus assembly. Lipid rafts are specialized membrane domains that float freely in the plasma membrane, and have a distinct composition of proteins and lipids (specifically sphingolipids and cholesterol) compared to the rest of the plasma membrane. In this paper, Gerl and colleagues quantified the lipid compositions for the host cell’s total membrane, the host cell’s apical membrane (where virus particles bud from), and influenza particles budded from these cells. The virus particles contained more sphingolipids and cholesterol than the host cell’s total or apical membrane, consistent with a model of virus budding from lipid rafts on the apical membrane. The electron micrographs above show purified spherical influenza virus particles recently budded from host cells.

Gerl, M., Sampaio, J., Urban, S., Kalvodova, L., Verbavatz, J., Binnington, B., Lindemann, D., Lingwood, C., Shevchenko, A., Schroeder, C., & Simons, K. (2012). Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane originally published in the Journal of Cell Biology, 196 (2), 213-221 DOI: 10.1083/jcb.201108175

November 17, 2011

A virus is both crafty and determined…pretty good for a non-living thing, right? Today’s image is from a paper discussing transport of the simian virus 40, which has some clever tricks up its little capsid sleeve to make the host cell help out with the viral infection.

Simian virus 40 (SV40) is a DNA virus that does not have its own membrane envelope, making transport into and around a cell a non-trivial task. SV40 first binds to a cell’s membrane, then uses endocytosis to enter the cell and travel to the endoplasmic reticulum (ER). From the ER, the virus penetrates the membrane to have access to the cytosol of the cell, where it can then reach the nucleus and replicate its genome. A recent paper describes this ER membrane penetration step, and reports that SV40 particles go through a major structural change within the lumen of the ER. According to Geiger and colleagues, this conformational change exposes a part of the minor viral protein VP2, which then embeds the virus into the ER membrane. This change attracts the cell’s own ERAD machinery, normally used to find and degrade misfolded proteins in the ER, which then allows the escape of the virus particles into the cytosol. Images above are electron micrographs of cells infected with SV40 for 2 (left), 6 (right), and 19 (bottom) hours. Early in the infection, virus particles are bound to endosomal or vesicle membranes (left, arrows). In the ER, the virus appears more compact (right, red arrows), indicating the structural change. Later, the ER membrane appears flattened around the virus particles (bottom, arrows).

Geiger, R., Andritschke, D., Friebe, S., Herzog, F., Luisoni, S., Heger, T., & Helenius, A. (2011). BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol Nature Cell Biology, 13 (11), 1305-1314 DOI: 10.1038/ncb2339
Adapted by permission from Macmillan Publishers Ltd, copyright ©2011

July 18, 2011

BAR domains are not just regions in a cell where proteins gather for a pint after a long day of cell division, migration, and sorting. No, BAR domains are regions on certain proteins that are able to bend membranes. This comes in handy for vesicle formation during endocytosis, and according to a recent paper, for plasma membrane organization.

Eisosomes are regions on the plasma membrane in budding yeast that are important for plasma membrane organization. They are positioned at the plasma membrane, where they sort membrane proteins and signaling molecules into small membrane invaginations. There is a long list of proteins found at eisosomes, many of which are uncharacterized. A recent paper looks at the functions of two core eisosome proteins – Pil1 and Lsp1. Olivera-Couto and colleagues found that these two proteins contain BAR domains, which are able to sense and change the curvature of membranes. Images above are electron micrographs of liposomes, which are artificially made vesicles. Untreated liposomes (top) are round, while vesicles treated with purified Pil1 or Lsp1 (middle and bottom) had tubules (arrows) deformed from the vesicles, showing that these proteins are capable of bending membranes.

Olivera-Couto, A., Grana, M., Harispe, L., & Aguilar, P. (2011). The eisosome core is composed of BAR domain proteins Molecular Biology of the Cell, 22 (13), 2360-2372 DOI: 10.1091/mbc.E10-12-1021

January 31, 2011

When many people think of membrane fusion, they often think of endocytosis and vesicle trafficking. Membrane fusion is a fascinating and dynamic process that is involved in so many processes in the cell, including nuclear envelope formation.

The nuclear envelope is composed of an inner and outer membrane that surrounds DNA and associated proteins. The nuclear envelope contains nuclear pores, which are channels that serve as sites of exchange between the nucleus and cytoplasm of the cell. The double membrane nature of the nucleus presents challenges in nuclear envelope and pore assembly that are overcome by membrane fusion events. A recent paper describes inner/outer membrane fusion during nuclear pore assembly and identifies an intermediate step in the process. By using cold temperatures that slow down nuclear pore formation in frog egg extracts, Fichtman and colleagues were able to clarify when and how fusion occurs between the inner and outer membranes. Specifically, the cold temperature treatment blocks inner/outer membrane fusion while allowing only the nuclear side of the pore to assemble. Images above show nuclear pore formation at room temperature (left set of images) and a cold temperature (right set of images). At room temperature, the nuclear membrane (green) and nuclear pores (red) form around chromatin (blue in merged images) after 15 minutes from the start of nuclear assembly. At cold temperatures, nuclear pores form more slowly and don’t appear on the nuclear envelope until 60 minutes.

Fichtman, B., Ramos, C., Rasala, B., Harel, A., & Forbes, D. (2010). Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly: Biochemical Demonstration and Molecular Analysis Molecular Biology of the Cell, 21 (23), 4197-4211 DOI: g/10.1091/mbc.E10-04-0309">10.1091/mbc.E10-04-0309