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Unlocking the Mysteries of Muscle Contraction: A Breakthrough in Ryanodine Receptor Structure

Ever wonder how our muscles work when we move? It all comes down to calcium—a key player, released from storage spaces within our muscle cells called the sarcoplasmic reticulum (SR), that regulates the muscle contraction. The crucial protein allowing calcium release is the type 1 ryanodine receptor (RyR1), an ion channel of enormous proportions (the largest known so far) that is embedded in the SR membrane.
RyR is more than just a protein; it’s a critical piece of the puzzle in understanding diseases like muscular dystrophy and heart failure. Plus, RyR serves as a potential pharmacological target, opening avenues for therapeutic interventions in muscle-related disorders.
Yet, working with such bulky proteins in its native state – within the cell membrane- is a challenging subject. Typically, the steps needed to resolve the structure of proteins normally involve using detergents and harsh conditions that disrupt the native state of a protein.
However, why is it crucial to comprehend the structure of proteins in their native state?? Because it enables us to mimic the conditions in living cells and gain valuable insights into their physiological relevance, their interaction with other molecules, exploring disease mechanisms, and facilitating drug development, among others.
So far, it has been used cryogenic electron microscopy (cryo-EM), an imaging technique that freezing samples and using electrons instead of light to reveal the protein’s intricate 3D structure (i.e., protein’s shape, how its various parts are arranged, and the interactions between its amino acids). While the 3D arrangement of the RyR1 protein had been solved at a very detailed level (referred to as the “High-resolution structure”), the configuration in its native state was still unknown.
In the pursuit to unravel the structure of proteins within native or near-native membranes, a recent breakthrough has provided a high-resolution view of RyR1.
Departing from traditional detergent-based methods used to solubilize and stabilize membrane proteins, Dr Melville Zephan, CUIMC postdoc, and colleagues adopted a gel-filtration approach with on-column detergent removal, they’ve captured RyR1 within liposomes—tiny bubbles mimicking cell membranes (Figure 1).

Figure1. Flow diagram illustrating protein-incorporated liposomes.
RyR1 from rabbit skeletal muscle was homogenized and purified using ion-exchange chromatography, a technique that separates compounds based on their electric charge. Liposomes, which are tiny vesicles made of self-assembly of lipid bilayers in water, were formed using gel filtration. This process helped incorporate RyR1 into liposomes and remove any excess or empty liposomes. (Adapted from Melville Z. Cell Press 2021)

This novel technique maintains a more natural environment, allows to purify membrane proteins and remove detergent molecules in excess, offering a clearer glimpse into how RyR1 functions (Figure 2).

Figure2. RyR1 representation in native and near-native membranes incorporated in liposome after gel filtration. (Adapted from Melville Z. Cell Press 2021)

Their study reveals how RyR1 behaves in its natural environment, forming a network of channels within cell membranes. While their investigation in liposomes gives only a partial view of this complexity (without seeing how molecules interact), it sets the stage for future investigations. This approach, extending beyond RyR, offers a blueprint for studying membrane proteins in native conditions.

In summary, this work successfully captured the intricate structure of RyR1 in liposomes opening doors to a better understanding of muscle physiology and offers a sharper lens for exploring the complex world of membrane proteins.

For more details on this research, refer to the article: “High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor”.

https://www.sciencedirect.com/science/article/pii/S0969212621002963?via%3Dihub

Reviewed by: Trang Nguyen, Carlos Diaz, Erin Cullen

 

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“super-SOX” hits iPSC research out of the park

The human body contains trillions of cells, which have extremely diverse functions. Remarkably, every one of these cells has the same genetic code. This diversity is possible because of the developmental process of differentiation. As an organism grows, cells take on traits that are useful in a particular context. For example, skin cells produce a protein called keratin that makes skin tough in order to protect the rest of the body from the outside world. Other cells don’t produce keratin because it would hinder their function. Once cells have differentiated and taken on a particular role, they can only generate more of the same kind of cell – skin cells only divide into more skin cells. This is because sections of DNA are hidden away during differentiation and stay hidden after the cell divides, ensuring that all the cells in a particular organ (like the skin) act like they’re supposed to.

To turn one type of differentiated cell into another was once thought to be impossible. The only cells that can generate any type of cell in the body are pluripotent stem cells (PSCs). PSCs have many potential applications in science, medicine, and industry, including disease modeling and drug testing as well as cell-based therapies and biotechnology. The opportunity to study PSCs can also enable scientists to answer basic questions about mammalian development. 

In 2006, Japanese researchers Takahashi and Yamanaka discovered that PSCs actually can be created from differentiated cells. Doing so requires just four key proteins: Oct4, Sox-2, Klf4, and cMyc. These proteins together are referred to as the Yamanaka factors. The discovery that the Yamanaka factors could turn a differentiated cell into a PSC was incredibly exciting to the scientific community, earning Dr. Shinya Yamanaka and a colleague the Nobel Prize in Physiology or Medicine in 2012.

Although this discovery created a broad array of new possibilities in science, it turns out that making PSCs in a laboratory is tough. Making healthy, high-quality PSCs from different species is even tougher. Induced pluripotent stem cells (iPSCs) have only been generated from a few species, and some cell lines create healthier iPSCs than others. A clearer understanding of how the Yamanaka factors interact with DNA and each other could reveal ways to make iPSC generation more robust and consistent, and realize the exciting potential of iPSCs. 

This challenge was taken on by a research group led by Drs. Sergiy Velychko and Caitlin MacCarthy in collaboration with CUIMC postdoc Dr. Vikas Malik and other teammates around the world. The group published a study this month in Cell Stem Cell describing a new protein based on the Yamanaka factor Sox2. They aptly call this protein “super-SOX” based on a number of studies demonstrating its ability to better generate iPSCs. The experiments also uncovered basic principles of pluripotency and development.

While three of the Yamanaka factors can be replaced with other proteins from the same family to generate iPSCs, Oct4 is the only member of its family (the POU family) that can induce pluripotency. The researchers set out to understand this oddity, with the goal of revealing the molecular interactions that lead to pluripotency. They first found that while a different member of the POU family could not generate iPSCs with the Yamanaka factor Sox2, it could generate them with a mutant protein based on another member of the SOX family, Sox17. 

Next, the researchers created a library to test different Sox2-Sox17 chimeras – mutant proteins that include components from both Sox2 and Sox17. Amino acids are the most basic building blocks of proteins, and surprisingly, the researchers found that a chimera with only one different amino acid from Sox2, Sox2AV, stabilizes the interaction between Sox2 and Oct4 and increases DNA binding efficiency. Oct4 is distinguishable from other members of the POU family by a negative charge in its “linker” domain. This negative charge allows Oct4 to form “salt bridges” connecting it to Sox2. Swapping the Sox2 amino acid alanine for the Sox17 amino acid valine promotes the formation of these bridges, encouraging Sox2AV and Oct4 to interact and bind to DNA together. The stronger connection between these two proteins dramatically improved pluripotency. 

Photo of healthy adult mice generated entirely from iPSCs and schematic of how outcomes change dramatically after replacing Sox2 with Sox2AV (Adapted from McCarthy et al., 2024).

The researchers hypothesized that the improvements in DNA binding they observed could promote the development of iPSCs into mature organisms. Live mice and rats have previously been generated from iPSCs, but these animals rarely survive to adulthood. Replacing Sox2 with Sox2AV – a simple change of one amino acid – when generating iPSCs dramatically increased mouse survival. 10/10 different cell lines treated with Oct4, Sox2AV, Klf4, and cMyc were able to produce live all-iPSC mouse pups, which only 3/8 cell lines treated with all the traditional Yamanaka factors were able to achieve. In the best-performing Sox2AV line, 43.3% of all-IPSC mice became healthy adults, compared to only 15.2% from the best-performing Sox2 line.

A further study found that a more complex chimera of Sox2 and Sox17 (Sox2-17) could improve iPSC generation in five different species: mice, cows, pigs, monkeys, and humans. These species were chosen for their potential to bring iPSC technologies to either biomedical research or industry – for example, iPSCs from livestock species could be key to producing lab-grown meat. Non-human primates and livestock species are less established in iPSC research, but the invention of Sox2-17, or “super-SOX”, opens many doors for future uses of iPSCs from these species. 

One barrier to creating iPSCs from human cells is age – differentiated cells from adults are highly resistant to becoming iPSCs. Strikingly, the group found that although the traditional Yamanaka factors could not produce iPSCs from skin cells of certain Parkinson’s disease patients, replacing SOX2 with the chimeric SOX2-17 could produce iPSCs from these samples.  Parkinson’s disease is characterized by the selective death of neurons in a brain region important for motor coordination called the substantia nigra, and could theoretically be treated by replacing lost neurons with iPSCs differentiated into neurons with a particular patient’s genetic code. The finding that SOX2-17 improves iPSC generation in cells from Parkinson’s disease patients demonstrates an exciting potential medical application for this discovery. 

The paper goes on to provide an explanation for these exciting findings. Prior to this study, it was known that although Oct4 is necessary for inducing pluripotency, too much Oct4 impairs iPSC generation. Free Oct4 encourages proliferation, which is actually detrimental to inducing pluripotency. Here, the researchers found that the stronger bond between Oct4 and Sox2AV or Sox2-17 lowers the proliferation rate, which improves the health of the cells that are generated. They went on to show that the bond between these two proteins is a main driver of the process of reverting a differentiated cell to a “naive” iPSC, which broadens the possibilities for the cell’s future development. 

This study makes a significant practical step toward realizing the potential of iPSCs. Simultaneously, it answers longstanding questions regarding how exactly the Yamanaka factors induce pluripotency, showing that the interaction between Oct4 and Sox2 has a central role. These findings have exciting implications for iPSC research, a field at the very forefront of science and medicine. 

Reviewed by: Trang Nguyen, Giulia Mezzadri, Carlos Diaz, Vikas Malik



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Take a look in the past to foresee the future: the evolution of genome engineering

Transposons, also known as mobile genetic elements, are segments of DNA capable of moving within the genome. This mobility can potentially induce mutations, as transposon “jumping” within a gene may interrupt the gene and trigger its loss. Transposons utilize their own enzymes, transposases, to move in and out of the genome.

Due to the potential harm caused by transposons, cells continually evolve strategies to control them. In response, transposons adapt, arming themselves with new strategies to evade cellular control. Mobilized transposons can hijack adjacent DNA information and transfer it to a new location, sometimes involving a gene that provides a survival advantage, thus increasing the transposon’s chances of propagation. Simultaneously, cells “steal” genes from transposons, repurposing them to function in processes advantageous to the cell. This ongoing arms race between transposons and host defense systems has facilitated a widespread exchange of genes between different life forms and is a major driver of genome evolution across all domains of life. Transposons play a crucial role in this process.

In bacteria, the CRISPR-Cas9 system is fundamental for protection against viral infections. Using a small RNA molecule called guide RNA, the CRISPR-Cas9 system acts as a nuclease, akin to DNA cutting scissors, destroying the viral genome. This system can be modified to edit genes in organisms beyond bacteria. The guide RNA is altered to direct the Cas DNA scissor to a gene of interest instead of a viral genome. This programmable gene-editing system has diverse applications, from basic biology research to disease treatments, and it earned the 2020 Nobel Prize in Chemistry for Emmanuelle Charpentier and Jennifer Doudna. Notably, scientists have recently traced the evolutionary origins of CRISPR-Cas9 to transposons.

In a recent study published in Nature, Columbia postdoc Chance Meers and his colleagues provided further insight into the evolution of the CRISPR-Cas9 nuclease system by examining how transposons use cellular nucleases to proliferate within genomes. They focused on a specific transposon system called insertion sequences (IS), which are bacterial transposons encoding only the genes necessary for transposon excision and movement to a new genomic location. This includes a transposase. However, many IS transposons contain an accessory gene that carries the information for a nuclease which functions similarly to the CRISPR-Cas system as an RNA-guided DNA cleaving enzyme.

The IS element does not need a nuclease for its movement, the transposase is sufficient to mobilize the transposon to a new location. So why are so many of the IS elements carrying those RNA-guided DNA scissors? While the mechanism by which IS elements are mobilized by their transposase is similar to a cut-and-paste process, leaving no copy at the original DNA location, Meers and colleagues discovered that the rate of excision is more efficient than the rate of integration. Without an additional system for transposon proliferation, the transposon would eventually be lost since not every excised copy manages to be reintegrated. The study of the authors revealed that the accessory CRISPR-like functioning nuclease guides a copy of the IS element back to its original location, generating two copies of the element—one at the original site and one at the newly inserted site. This changes the mechanism from cut and paste to cut and copy when the transposase function is complemented by the accessory nuclease, increasing the transposon copy number present in the genome, and thus serving for the transposon selfish proliferation (Figure 1).

Figure 1. RNA-guided nucleases assist transposon survival via guiding specific breaks at donor sites and transposon restoring repair. During DNA replication the two strands of the DNA duplex are separated and each strand serves as a template for the synthesis of a new DNA molecule. Single stranded DNA facilitates the transposition mediated by the transposase of IS elements. The excision of the transposon leads to its loss at one of two DNA strands, while DNA replication restores the transposon on the other DNA strand. Simultaneously, transposon excision restores a target site at the transposon donor location which is specifically recognized by a guide RNA (blue segment). This guide RNA directs the accessory nuclease on the excision location and DNA cleavage occurs. The resulting double strand break (DSB) is lethal for the bacteria and its only chance of survival is to copy the information from the homologous newly synthesized intact DNA molecule that contains the transposon. This leads to reconstitution of the transposon at the donor site on the two newly synthesized DNA duplexes. Transposition at the new target sites will produce new guides, specific for the new insertion site location (orange segment), which will facilitate the future transposon spread and maintenance by identical mechanism. 

By developing powerful assays to track the movement of transposons within bacterial genomes, or other small DNA molecules called plasmids, as well as from one bacterium to another, Meers and his colleagues uncovered how those RNA-guided DNA cutting nucleases work. The authors’ discovery enhances our understanding of how proteins collaborate with RNA guides to target and edit genomes. In addition, the study unveils the original role of CRISPR’s nucleases from an evolutionary perspective before they were repurposed by the bacterial host genome to fight viruses, which was to serve the selfish propagation of transposons. Moreover, given the abundance of transposons in genomes, it is highly likely that other systems different from CRISPR-Cas9 and derived from transposon genes exist, waiting to be discovered and potentially harnessed, expanding the biotechnological tools available for programmable and specific genome engineering. Thousands of ancient transposons in bacterial genomes carry RNA-guided DNA nucleases that can potentially be programmed to cut DNA similarly to the CRISPR-Cas9 system.

Reviewed by: Trang Nguyen, Giulia Mezzadri

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When Numbers Play Tricks: Unraveling The Brain’s Biases

Imagine living in a dark cave, with your entire understanding of the world based on shadows on the wall. Sounds unrealistic and terrifying, right? However, this allegory presented by Plato is an apt metaphor for our brain’s perception of the real world. While we might believe we perceive reality in its entirety, our brain can only provide a shadowy representation of the variables in our environment, and our decisions are based on these shadowy representations.

A comforting thought might be that numbers are a universal language for Western countries and, specifically, for those who use Arabic numerals. For instance, when the price of a good is marked as $14, it conveys an unambiguous and specific value, meaning that one would unequivocally expect to pay exactly that amount. However, experiments show that people make mistakes when dealing with Arabic numerals. For instance, under time constraints, the closer two numbers are in value, the more challenging it becomes for us to rapidly and accurately pinpoint the larger one. These mistakes are very similar to those made in psychophysics tasks involving physical stimuli, such as comparing the length of segments or averaging the orientation of tilted lines. These results, together with neurobiological studies, suggest the existence of a representation system for numbers that is similar to how we interpret physical stimuli.

A popular idea in theoretical neuroscience is that while the brain’s computational abilities have inherent limits, leading to imprecise representations, these representations are optimal within those constraints. This theory, called “efficient coding”, suggests that our brain’s perceptions are influenced by how often these magnitudes are encountered (i.e., the prior). For example, vertical and horizontal orientations are perceived with more clarity than oblique ones, likely because they’re more common in our environment.

A recent study (https://www.nature.com/articles/s41562-022-01352-4), led by Columbia postdoc Arthur Prat-Carrabin and published in Nature Human Behaviour, delves into whether our brain treats numbers the same way it treats physical stimuli. In their experiment, participants were asked to determine which series of numbers, red or green, had a higher average value. For instance, as shown in Figure 1, the number 79.60 would flash in red on the computer screen, followed by 44.92 in green, and so forth. Participants were tasked with rapidly and intuitively calculating the average of the red and green numbers to determine which sequence had the greater average. To investigate how the frequency at which numbers were encountered impacts their representation, numbers were drawn from different distributions: one, in which smaller numbers were more frequent (Downward prior); another, in which all numbers had equal chances (Uniform prior); and a third, in which larger numbers were more frequent (Upward prior).

Figure 1: Example trial of the task. Participants were presented with 5 red and 5 green numbers and had to choose the color with the higher average.

To analyze the participants’ decisions, the researchers compared their answers with multiple computational models characterized by two components: first, whether or not the numbers are encoded with a bias that depends on the value of the number, and second, whether or not the imprecision (the noise) with which numbers are represented varies with the number. Their results showed that the model that best aligned with the participants’ answers had to include both components. Notably, less common numbers are fuzzier in their perception. So, when using the Downward prior, bigger numbers are encoded with more noise, while with the Upward prior, the smaller numbers are the noisiest ones.

This discovery not only supports the “efficient coding” theory, which posits that the brain encodes and represents information in the most efficient way possible, but also showcases its applicability beyond just physical perceptions. Whether we’re assessing the speed of a car, the talent of a dancer, or the sweetness of a cake, our brain might use a universal mechanism to represent these variables. This mechanism dynamically adjusts to the statistical distribution of numbers that are expected or experienced, which implies that our understanding of numbers and magnitudes isn’t static but can be influenced by our prior experiences and expectations. In the near future, we might be able to design environments that help people refine their perceptions (such as by crafting digital games to enhance the consumer’s responsiveness to certain prices), allowing them to better discern specific value ranges and improve their decision-making.

Reviewed by: Emily Hokett, Trang Nguyen, Martina Proietti Onori

 

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Masters of disguise: unlocking the secrets of color-changing cuttlefish

Cuttlefish, octopus, and squid are fascinating marine creatures that have perfected the art of camouflage underwater. With uncanny speed, these soft-bodied cephalopods can change the color, pattern, and texture of their skin to blend seamlessly into their surroundings, impressively adapting to their environment. Unlike many animals, cuttlefish don’t rely on fur or feathers to hide in the background. Instead, they actively manipulate thousands of pigment cells in their skin to acquire the color of the environment around them. This intricate disguise process starts in their brains, as camouflage is a response to the animal’s perception of the external world. To conceal their bodies, cephalopods convert visual inputs into neural representations within their brain, ultimately transmitting signals all the way to the skin, where thousands of tiny structures called chromatophores adjust to allow color changes. However, camouflage is just one captivating aspect of cuttlefish biology. These marine animals present a rich repertoire of signaling behaviors for mating and communication and they are proficient learners, with memory capabilities not often seen in invertebrates. Collectively, these attributes position cephalopods as extraordinarily captivating subjects within the realm of biological studies.

Researchers at Columbia University’s Zuckerman Institute, led by Dr. Richard Axel, have made significant progress in comprehending the shape-shifting abilities of cuttlefish. In a recent article published in Current Biology, they generated a detailed neuroanatomical brain map, revealing insights into how their skin transformation is controlled. Tessa Montague, PhD and colleagues focused on the dwarf cuttlefish (Sepia bandensis), a small tropical species found around coral reefs in the Indo-Pacific Ocean (Figure 1A). The dwarf cuttlefish is an intriguing model for neuroscience research with its rich array of dynamic social behaviors and camouflage. Through an advanced imaging technique called MRI (magnetic resonance imaging), computer programming and web design they constructed a 3D atlas illustrating the dwarf cuttlefish’s brain anatomy (Figure 1B).

Figure 1. (A) Adult dwarf cuttlefish. (B) 3D template brain, based on magnetic resonance imaging (MRI) of 8 cuttlefish brains, (i) anterior view, (ii) right view, and (iii) posterior view. The abbreviations in color indicate the different brain structures that were identified and mapped.  Modified from T. G. Montague et al., 2023

By scanning the bodies and brains of male and female cuttlefish, the researchers identified 32 distinct lobes or functional units within the cuttlefish brain (Figure 1B). Each lobe is densely packed with neurons and performs specialized tasks. The two largest lobes, making up 75% of the total brain volume, are the optic lobes (O in Figure 1B). They receive direct projections from the eyes and process visual information, a crucial step in enabling cuttlefish camouflage. Notably, other key lobes in the camouflage pathway include those controlling the chromatophores, the pigment-filled saccules in cuttlefish skin that provide the color. When the lobes send signals to the chromatophores, these rapidly expand or contract to alter skin shades on a millisecond timescale. The lateral basal lobe (LB in Figure 1B) for example, is the lobe involved in establishing the most appropriate skin pattern components for camouflage. Another brain area highlighted by the atlas is the vertical lobe complex (V and surrounding lobes in Figure 1B), which previous studies suggest plays a key role in learning and memory. Unlocking the functions of this lobe could reveal the neural basis for complex behaviors like camouflage.

While past studies have mapped the brains of related cephalopods like squid and octopus, this is the first complete atlas for a cuttlefish species and it provides the neuroscience field with a valuable comparative perspective.  The researchers found strong similarities in the anatomy of the dwarf cuttlefish with the common cuttlefish, despite differences in size and camouflage strategies between the species. This suggests that fundamental aspects of brain organization are conserved, at least among close cephalopod relatives. It also highlights how flexible cuttlefish brains are: they can generate very different camouflage patterns using essentially the same basic circuit layout. Exploring what accounts for such flexibility on the microscopic scale will be the next challenge.

To maximize the utility of the brain atlas, for both educational and research purposes, the authors built an interactive freely available web tool, called Cuttlebase, where users can identify specific brain regions in the histological 2D atlas (that shows the cellular components in the cuttlefish brain)  and use the 3D model of the brain to explore the different lobes (Figure 2).

Figure 2. Cuttlebase is an interactive scientific web toolkit for the dwarf cuttlefish that includes a histological brain atlas in 2D and a 3D body and brain atlas to visualize and explore the different organs and brain lobes. Obtained and modified from https://www.cuttlebase.org/

This atlas serves as an invaluable tool for the scientific community to explore the basic anatomical components of complex behaviors and can give us insight into how brains are capable of representing information. It also offers an invaluable anatomy lesson, giving scientists a privileged peek inside the ingenious brains of these undersea masters of disguise. With a detailed understanding of their sophisticated neural systems, unraveling cuttlefish mysteries seems closer than ever.

Reviewed by: Trang Nguyen, Maaike Schilperoort

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Copy & Paste – an essential mechanism to repair damaged DNA

Maintaining genome integrity is crucial for cell survival and genome instability is one of the hallmarks of cancer. The genomes of many living organisms including humans are composed of different numbers of DNA molecules that are folded into structures called chromosomes. Each chromosome represents one double stranded DNA molecule. Most of our cells harbor two sets of each chromosome, which are denoted as homologous chromosomes (Figure 1, left panel). The homologous chromosome pairs can contain identical or different versions (alleles) of the genes that they carry since they are inherited from each parent. In this sense homologous chromosomes are genetically non-identical.

Most of the cells in our body are constantly renewed and replaced in a process that involves cellular division. During cell division the genome needs to be duplicated so that each of the newly formed daughter cells receives a genome copy. The process of DNA duplication is called DNA replication. During replication, each of the two strands of the DNA double helix serves as a template for the synthesis of a new complementary DNA strand. These chromosome copies that are produced during cell division are named sister chromatids (Figure 1, right panel), and they are found transiently in the cells just before the division. Unlike the homologous chromosomes, the alleles of the genes in the sister chromatids are genetically identical since they are produced by DNA replication.

Figure 1: Schematic representation of the homologous chromosomes and the sister chromatids produced during cellular division. Created with BioRender.com

During DNA replications, as well as under the influence of diverse environmental and endogenous agents, lesions constantly occur on DNA. These DNA “injuries” can trigger mutations, compromise the genome integrity, or even cause cell death. DNA double-strand breaks (DSBs) are one of the most deleterious types of DNA lesions that can lead to gross chromosome rearrangements. Similar rearrangements are very common in cancer.  One of the major mechanisms for DNA DSBs repair is homologous recombination (HR), a process explained below. Mutations in different genes from the HR pathway have been associated with diseases like cancer of Fanconi anemia, as well as to hypersensitivity to DNA-damaging agents which increase mutation accumulation.

One of the versions of HR involves the use of the intact sister chromatid as donor of information. Since the sister chromatid has an identical sequence to the damaged DNA molecule, this repair system faithfully restores the genetic information and is considered as being error free. Homologous recombination can also occur between the homologous chromosome pairs in the case of non-dividing cells. In HR, the DNA DSB ends are processed and a long single-stranded DNA (ssDNA) overhang is left to serve as platform for the assembly of the protein machinery mediating the repair (Figure 1, a). This ssDNA is coated by the protein RPA to prevent degradation and folding. Subsequently RPA is replaced by RAD51, into a structure termed the presynaptic nucleoprotein filament. This filament is capable of searching for a homologous undamaged DNA molecule that will serve as a template for repair. This exchange between RPA and RAD51 on ssDNA in the presynaptic filament assembly is facilitated by mediators of HR. One of those mediators is a complex of four proteins that are paralogues of RAD51: the BCDX2 complex. However, the exact mechanism of action of the BCDX2 complex in HR remained elusive.

Figure 2. Double-strand breaks (DSBs) can be repaired by several homologous recombination (HR)-mediated pathways. Represented is a simplified version of the “synthesis-dependent strand annealing HR pathway”. In all pathways, the repair is initiated by resection of a DSB to provide 3′ single-stranded DNA (ssDNA) overhangs (a and b). The ssRNA is rapidly coated with RPA to prevent damage and folding (c). RPA is exchanged with RAD51 with the help of HR mediator complexes, which are composed from different RAD51 paralogues, one of which is four protein complex BCDX2 (d). RAD51 coated DNA can search for the non-damaged homologous chromosome / sister chromatid pair and invade that intact DNA duplex (e). After strand invasion and “copying” of the information from the intact DNA on one of the damaged strands from the blue DNA duplex, the reaction can proceed to hybridization to the ssDNA on the other break end of the blue DNA duplex, followed by DNA synthesis (f) and restoring  of blue DNA molecule (g). Created with BioRender.com

Recent studies from Columbia postdoc Aviv Meir and colleagues revealed the structure of the human BCDX2 complex. Cryogenic electron microscopy, a technique that allows the high-resolution structure determination of biomolecules in solution, was used to resolve both the  free and single-strand DNA-bound states of BCDX2. This provided the first structural information of one of the RAD51 paralogues complexes. This structural information provides insight into how the complex assembles and disassembles, which in turn is linked to the regulation of its function. The scientists also discovered by single molecule analysis that the association of BCDX2 with RPA–ssDNA enhances the rate of assembly of the RAD51–ssDNA filament. In humans, BCDX2 binds the RPA–ssDNA prior to the arrival of RAD51 and then promotes the RAD51 filament assembly. This novel mode of action for the proteins of the BCDX2 complex is different from what was previously observed in other organisms, where BCDX2 only transiently associates with the ssDNA. The work by Dr. Meir and colleagues, recently published in the prestigious journal Nature, not only elucidates how BCDX2 mediates RPA–RAD51 exchange on ssDNA but also provides a foundation for deciphering how alterations in BCDX2 subunits that were found in patients with cancer can impact genome repair and can lead to the pathogenesis. This valuable information opens the doors for future targeting of those “defectuous” BCDX2 parts for therapeutic developments.

Reviewed by: Maaike Schilperoort, Apurva Limaye, Giulia Mezzadri , Trang Nguyen

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Lactic acid – a new energy fuel source in brain tumor

What does lactic acid do to the body?

Lactic acid is produced when the body breaks down carbohydrates in low oxygen levels to generate energy. It is mainly found in muscle cells and red blood cells. An example of lactic production is when we perform intense exercise. 

Glucose, glutamine, fatty acids, and amino acids are well-known energy sources for cell growth and division. In the past, lactic acid has been known as a by-product of glycolysis, a process in which glucose is broken down through several enzyme reactions without the involvement of oxygen. However, recent studies showed that lactic acid is a key player in cancer cells to regulate tumor cell growth and division, blood vessel formation, and invasion. The tumor cells prefer to use glycolysis to produce energy and lactic acid despite the abundance of oxygen levels. Lactic acid is an alternative fuel source for glucose-deprived tumors to avoid cell death.

Lactic acid is transported through the membrane via the monocarboxylate transporter 1 (MCT1). A research group at Columbia University led by Dr. Markus Siegelin in the department of Pathology and Cell Biology showed a substantial presence of lactic acid in the citric acid cycle (TCA cycle), a series of chemical reactions to generate energy, in the glioblastoma cells cultured in the nutrient deprivation condition (low glucose and glutamine concentration). When the glucose and/or glutamine concentrations increased, less lactic acid was involved in the TCA-cycle metabolites. The uptaken lactic acid in the TCA-cycle was traced by using a method called C13 carbon tracing and was analyzed by liquid chromatography-mass spectrometry to identify the structure of different molecules. The researchers concluded that lactic acid is used as a fuel source to generate the energy in the brain tumor cells. Furthermore, lactic acid is converted to Actetyl-CoA and contributed to the gene modification in glioblastoma cells (Figure 1). These novel findings were published in a prestigious journal,  Molecular Cell. 

Figure 1: Role of lactic acid in the epigenetic modification of glioblastoma cells. Lactic acid is transported to the membrane via the monocarboxylate transporter 1 (MCT1) and contributed to the TCA cycle as a fuel source to generate the energy. Lactic acid is converted to Actetyl-CoA and contributed to the gene modification in glioblastoma cells. Suppressing the TCA cycle by using the targeted drug, namely CPI-613 (devimistat) leads to the abrogation of lactic acid in the energy production. The figure was generated by Biorender.

From these findings, the authors proposed to use CPI-613 (devimistat) drug, which targets TCA-cycle metabolites (Figure 1), to  treat glioblastoma cells. Indeed, CPI-613 showed a suppression of cellular viability in vitro of glioblastoma cells and an extension of the animal survival curve in the mouse model. The authors suggested that the combination of CPI-613 with other standard care treatment in glioblastoma such as temozolomide and radiation could be a potential clinical therapy for patients with glioblastoma.

Read more about this exciting finding here:

https://www.sciencedirect.com/science/article/pii/S1097276522006475 

Reviewed by: Pei-Yin Shih, Sam Rossano, Emily Hokett

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A small investment could reap a big reward

It is well-established that children growing up in economically disadvantaged circumstances can experience a wide variety of challenges to their development. For example, in the domain of language, psychological research shows that even before children reach the age of 3, those from lower socioeconomic (SES) backgrounds hear fewer and less complex words from caregivers compared to their more advantaged peers. This phenomenon places these children at risk of later language learning difficulties. Research groups and other stakeholders around the globe are working tirelessly to craft intervention programs, in a variety of contexts, to target the optimal development of children from under-resourced environments. To target language outcomes, for instance, an initiative called Talk With Me Baby aims to help parents learn about and engage in crucial linguistic interactions with their children from their earliest days.

One prominent intervention in this field is taking a different approach. The Baby’s First Years (BFY) project, directed by a team of leaders in the realms of neuroscience, psychology, and economics, provides families living in poverty with cash and is studying how this money might impact developmental outcomes. These scientists recruited 1,000 mothers from around the US just hours after their babies were born. Half of the mothers are assigned to a high-cash gift group ($333 per month) and the other half are assigned to a low-cash gift group ($20 per month). These payments come in the form of a debit card and the moms are allowed to spend these funds however they’d like.

The “4 My Baby” card, the debit card that BFY mothers receive. Courtesy of Baby’s First Years.

After one year of these payments, the researchers went into the homes of the families and measured a variety of both maternal and child outcomes. One of these measures was EEG, or electroencephalography, to capture the brain activity of the infants. EEG measures the electrical activity occurring between cells in the brain. The findings, published in a recent paper in PNAS by lead author and Columbia postdoc Dr. Sonya Troller-Renfree, captured global attention. She and her team found that infants whose mothers were assigned to the high-cash gift group displayed more high-frequency brain activity compared to children in the low-cash group. Importantly, previous research links this high-frequency activity to the development of thinking and learning. Although the evidence is not air tight, Dr. Troller-Renfree and her team are the first to show there may be a causal link between poverty reduction and changes in brain activity.

There are many pathways through which the gifted money might be impacting children’s brains, which is currently under investigation. It is important to understand that this study is still ongoing and the children in the sample are all currently around 3-years-old. The research team plans to assess the mothers and preschoolers at age 4 on a variety of outcomes. This finding on the potential link between poverty reduction and the brain, along with other work demonstrating that economic support for parents could greatly benefit children’s outcomes, has important implications for public policies that support families and children. While the US has a long way to go in supporting its youth, growing evidence indeed supports the idea that relatively minor investments in children can positively impact their trajectory.

Dr. Sonya V. Troller-Renfree is a Goldberg Postdoctoral Fellow in the Neurocognition, Early Experience and Development Lab at Teachers College, Columbia University. Her research focuses on the effects of early adversity and poverty on cognitive and neural development. She intends to continue examining these questions as part of her new, federally-funded Pathway to Independence Award (K99/00). You can stay up-to-date on her research findings on Twitter at @STRscience or on her website: www.sonyatrollerrenfree.com.

 

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Covid-19 and Immunity after Organ Transplant

Over the last two years, the SARS-CoV-2 (Covid-19) pandemic has been at the forefront of media coverage. Hospitals have been overwhelmed, full cities locked down, travel banned, and we are all desperately waiting for a return to the normalcy that immunity promises. However, the development and retention of immunity can be dependent on the individual, and Covid-19 has been particularly daunting to individuals  with weakened immune systems (people who are immunocompromised). These individuals are at an increased risk of succumbing to Covid-19. Overall, it has been easy to identify the individuals that fall into this risk category. However, there has been limited research on the immunity of individuals that  have undergone organ transplants. In a new article by Dr. Mithil Soni, researchers have identified the effects of a solid organ transplant (SOT) on the development and retention of immunity to a plethora of viruses including SARS-CoV-2. SOTs are transplants that  include the kidney, liver, heart, lungs, intestines, and pancreas .

 Dr. Soni and colleagues also focus on the immunity generated by T cells, or cells beyond antibodies that play a role in killing viruses that enter the body. In this study, they focused on the immune response of one patient, a 33-year old male  suffering from erythropoietic protoporphyria, a genetic metabolic disorder that results in excessive liver damage. This subject underwent a SOT and received a liver. When undergoing a SOT, individuals are usually put through a stringent course of immunosuppressants to prevent organ rejection, which places them in the category of immunocompromised.  To their surprise, during a check-up this patient was found to have antibodies for SARS-CoV-2, indicating a previous Covid-19 infection without any serious symptoms. The ability to overcome Covid-19 with minimal symptoms while being classified as immunocompromised intrigued Dr. Soni and colleagues, and the patient agreed to provide his blood for further testing.  

The team went on to test the patient’s immune response to many infections that usually impact immunocompromised individuals. They tested the blood’s immune response to cytomegalovirus and BK virus, two viral infections that immunocompromised and SOT patients are prone to. They also tested the response to Epstein-Barr virus, which can cause Mononucleosis. From the blood, Dr. Soni and colleagues were able to collect and grow the T cells in their lab, exposed them to viruses, and measured their release of cytokines, proteins that are important for a strong immune response. They found a very strong T cell immune response against both cytomegalovirus and BK virus. They also tested the immune response to SARS CoV-2 and other coronaviruses and found a similar level T cell immune response as seen with cytomegalovirus and BK virus. 

These findings overall indicated that the SOT patient continued to have a robust immune response to multiple viruses despite the immunocompromised status. This study shows that it is possible to have robust immune responses to viruses including SARS CoV-2 in an immunocompromised state such as seen after a SOT. However, this research is based on a single case study. To truly understand T-cell memory and activity in immunocompromised individuals much more research has to be done. This means Dr. Soni and colleagues still have their work cut out for them and are actively expanding the research done here. Their next immediate steps are to repeat this study with blood from a larger group of healthy and immunocompromised individuals in the hopes that they will eventually be able to answer the question of SOT and immunity.

Figure: Depiction of increased immunity after SOT.  Top: Liver transplant. Bottom: Expected T cell activity in response to virus vs actual T cell activity in response to virus. 

 

Dr. Mithil Soni, is a previous Postdoctoral Research Fellow and current Associate Research Scientist at Columbia University.

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Let’s get MDM2 and MDMX out of the shadow of p53

When it comes to cancer, one molecule stands out as being among the most extensively studied: the p53 tumor suppressor protein. p53 can exist in cells in several different forms. When p53 is in its so-called wild-type form, it is capable of activating various responses that contribute to tumor suppression. In their recent review, Columbia postdoc Rafaela Muniz de Quieroz and colleagues summarize the vast scientific literature on two key regulators of p53: MDM2 and MDMX. Both MDM2 and MDMX are known to interact with p53 and disrupt its function. Their absence has been linked not only to increased cancer development, but also to a number of dysfunctions, including embryonic lethality in mice. MDM2 has been shown to negatively regulate p53 by diverse mechanisms spanning from expression of the p53 gene to degradation of the p53 protein or its expulsion from the cellular nucleus, where the protein accomplishes its function. Although very similar to MDM2, MDMX is less well studied. We do know, however, that MDMX is a protein that can work together with the MDM2  in p53 degradation.

While many reviews and studies have pointed to the roles of MDM2, and to a lesser extent of MDMX, in p53 regulation, the current review by Quieroz and her colleagues  puts a larger focus on the myriad of p53-independent activities of MDM2 and MDMX. The authors provide important details about the p53-independent functions of both MDMX alone and as part of a MDM2–MDMX complex. The review discusses some key features in the structure and function of the proteins, including  key parts  that are relevant for their function, for some associated abnormalities, or for the formation of MDM2 and MDMX complexes.

MDM2 and MDMX are regulated on multiple levels within cells. These include regulation on the DNA level, including usage of several alternative promoters (DNA sequences needed to turn a gene on or off). One of the promoters of MDM2 and MDMX is regulated by their target p53, but there are also p53-independent promoters capable of switching on the genes of MDM2 and MDMS regardless of p53. In addition, numerous variations in the DNA sequences, the so-called single nucleotide polymorphisms (SNPs), affect the expression of the two genes and are relevant to different pathologies. Regulation on the RNA level includes co-transcriptional regulation like alternative splicing, as well as post-transcriptional regulation by microRNAs, long non-coding RNAs, circular RNAs, or RNA binding proteins. The review also presents a detailed characterization of the regulation of MDM2 and MDMX at the protein level, by summarizing data on numerous post-translational modifications or interacting partners of the two proteins, with regards to the different p53 contexts of the cited studies. Amongst the presented binding partners are some of the more recently identified interactors of the MDMs, which include proteins involved in the defense against several viruses. Overall, both MDM2 and MDMX stand out as extensively regulated at virtually every known level which according to the authors “attests to their relevance not only as inhibitors of p53 but of myriad other cellular activities and outcomes on their own”.

Since MDM2 and MDMX have majorly been studied in their relation to inhibit wild-type p53, of a particular interest stands a section of the review summarizing numerous processes in which the two proteins have been shown to be involved in cells lacking wild-type p53 (Figure 1).

Figure 1: Nonmalignant disease (left) and cancer-related (right) p53-independent functions of MDM2 and MDMX (adapted from Figure 4 of the review).

As shown in Figure 1, the p53-independent roles of MDM2 and MDMX in cancer and in other pathologies are versatile. That hints to the importance of uncovering molecules that can modulate the deleterious effects associated with dysfunctions of the two MDMs. A summary of numerous molecules that were shown to regulate the two proteins and thus consist of potential therapeutic targets, are also discussed in the review. Again the authors put an emphasis on how such small molecules might be useful in cells that lack wild-type p53. This is important not only because the two proteins have multiple functions other than regulating wild-type p53 which can be studied in such cells, but also because an important percentage of tumors is characterized by absence of wild-type p53.

The last section of the review points out some outstanding questions and directions for future research. If the fascinating questions of the versatile p53-independent roles MDM2 and MDMX have sparked your interest, find out more from the original paper.

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