Compared to bacteria, in eukaryotes the genetic material is located in the cell nucleus. Its outer shell consists of the nuclear membrane with numerous nuclear pores. Molecules are transported into or out of the cell nucleus via these pores. Beneath the membrane lies the nuclear lamina, a threadlike meshwork merely a few millionths of a millimeter thick. This stabilizes the cell nucleus and protects the DNA underneath from external influences. Moreover, the lamina plays a key role in essential processes in the cell nucleus -- such as the organization of the chromosomes, gene activity and the duplication of genetic material before cell division.

Detailed 3D image of the nuclear lamina in its native environment

Now, for the first time, a team of researchers headed by cell biology professor Ohad Medalia from the Department of Biochemistry at UZH has succeeded in elucidating the molecular architecture of the nuclear lamina in mammalian cells in detail. The scientists studied fibroblast cells of mice using cryo-electron tomography. "This technique combines electron microscopy and tomography, and enables cell structures to be displayed in 3D in a quasi-natural state," explains Yagmur Turgay, the first author of the study. The cells are shock-frozen in liquid ethane at minus 190 degrees without being pretreated with harmful chemicals, thereby preserving the cell structures in their original state.

"The lamin meshwork is a layer that's around 14 nanometers thick, located directly beneath the pore complexes of the nuclear membrane and consists of regions that are packed more or less densely," says Yagmur Turgay, describing the architecture of the nucleoskeleton. The scaffold is made of thin, threadlike structures that differ in length -- the lamin filaments. Only 3.5 nanometers thick, the lamin filaments are much thinner and more delicate than the structures forming the cytoskeleton outside the cell nucleus in higher organisms.

New approach for research on progeria and muscular dystrophy

The building blocks of the filaments are two proteins -- type A and B lamin proteins -- which assemble into polymers. They consist of a long stem and a globular domain, much like a pin with a head. Individual mutations in the lamin gene elicit severe diseases with symptoms such as premature aging (progeria), muscle wasting (muscular dystrophy), lipodystrophy and damage of the nervous system (neuropathies). "Cryo-electron tomography will enable us to study the structural differences in the nuclear lamina in healthy people and in patients with mutations in the lamin gene in detail in the future," concludes Ohad Medalia. According to the structural biologist, this method permits the development of new disease models at molecular level, which paves the way for new therapeutic interventions.

Story Source:

Materials provided by University of Zurich. Note: Content may be edited for style and length.

Read more from the original source:
Molecular structure of the cell nucleoskeleton revealed for the first time - Science Daily

Working with yeast and human cells, researchers at Johns Hopkins say they have discovered an unexpected route for cells to eliminate protein clumps that may sometimes be the molecular equivalent of throwing too much or the wrong trash into the garbage disposal. Their finding, they say, could help explain part of what goes awry in the progression of such neurodegenerative diseases as Parkinson's and Alzheimer's.

Proteins in the cell that are damaged or folded incorrectly tend to form clumps or aggregates, which have been thought to dissolve gradually in a cell's cytoplasm or nucleus thanks to an enzyme complex called the proteasome, or in a digestive organelle called the lysosome.

But in experiments on yeast, which has many structures similar to those in human cells, the Johns Hopkins scientists unexpectedly found that many of those protein clumps break down in the cell's energy-producing powerhouses, called mitochondria. They also found that too many misfolded proteins can clog up and damage this vital structure.

The team's findings, described March 1 in Nature, could help explain why protein clumping and mitochondrial deterioration are both hallmarks of neurodegenerative diseases.

Rong Li, Ph.D., professor of cell biology, biomedical engineering and oncology at the Johns Hopkins University School of Medicine and a member of the Johns Hopkins Kimmel Cancer Center, who led the study, likens the disposal system to the interplay between a household's trash and a garbage disposal in the kitchen sink. The disposal is handy and helps keep the house free of food scraps, but the danger is that with too much trash, especially tough-to-grind garbage, the system could get clogged up or break down.

In a previous study, Li and her team found protein aggregates, which form abundantly under stressful conditions, such as intense heat, stuck to the outer surface of mitochondria. In this study, they found the aggregates bind to proteins that form the pores mitochondria normally use to import proteins needed to build this organelle. If these pores are damaged by mutations, then aggregates cannot be dissolved, the researchers report. These observations led the team to hypothesize that misfolded proteins in the aggregates are pulled into mitochondria for disposal, much like food scraps dropped into the garbage disposal. Testing this hypothesis was tricky, Li says, because most of the misfolded proteins started out in the cytoplasm, and most of those that enter mitochondria quickly get ground up.

As a consequence, Li and her team used a technique in which a fluorescent protein was split into two parts. Then, they put one part inside the mitochondria and linked the other part with a misfolded and clumping protein in the cytoplasm. If the misfolded protein entered the mitochondria, the two parts of the fluorescent protein could come together and light up the mitochondria. This was indeed what happened.

"With any experiment," Li says, "you have a hypothesis, but in your head, you may be skeptical, so seeing the bright mitochondria was an enlightening moment."

To see what might happen in a diseased system, the team then put into yeast cells a protein implicated in the neurodegenerative disease known as amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease. After a heat treatment that caused the ALS protein to misfold, it also wound up in the mitochondria. The researchers then did an experiment in which a lot of proteins in the cytoplasm were made to misfold and found that when too much of these proteins entered mitochondria, they started to break down.

The team wanted to make sure that the phenomenon it had observed in the yeast cells could also happen in human cells, so the scientists used the same split-fluorescent protein method to observe misfolded proteins to enter the mitochondria of lab-grown human retinal pigmented epithelial cells. As observed in yeast, misfolded proteins, but not those that were properly folded, entered and lit up mitochondria.

Biological systems are in general quite robust, but there are also some Achilles' heels that may be disease prone, Li says, and relying on the mitochondrial system to help with cleanup may be one such example. While young and healthy mitochondria may be fully up to the task, aged mitochondria or those overwhelmed by too much cleanup in troubled cells may suffer damage, which could then impair many of their other vital functions.

Story Source:

Materials provided by Johns Hopkins Medicine. Note: Content may be edited for style and length.

View original post here:
In cleaning up misfolded proteins, cell powerhouses can break down - Science Daily

Professor David R. Greaves, University of Oxford, UK, discusses the technology that is enabling him to research tissue repair and chronic inflammation in real time

David R. Greaves,University of Oxford, UK

Professor David R. Greaves is using the latest live cell imaging technology to carry out ground-breaking research into inflammation biology. Sonia Nicholas, Associate Editor for SelectScience, spoke to Professor Greaves to find out more.

SN: Please could you confirm your name, full job title and place of work.

DRG: Im Professor David R. Greaves, I am a University Lecturer in Cellular Pathology at the Sir William Dunn School of Pathology, University of Oxford, UK.

SN: Could you tell us about your job, what you do and what your responsibilities are?

DRG: I run a research laboratory working on macrophage biology and inflammation. I am very interested in macrophage chemotaxis as well as other aspects of macrophage cell biology such as phagocytosis and cytokine secretion.

In addition to doing biomedical research I run the BM Principles of Pathology course for second year medical students at the University of Oxford, I run a final year lecture course in Inflammation Biology, I run the British Heart Foundation 4-year Cardiovascular Sciences PhD program and I am a Tutorial Fellow in Medicine at Hertford College where I give tutorials in Biochemistry, Cell Biology, Endocrinology, Medical Genetics and Pathology.

Inflammation and disease

SN: Can you tell us more about your research into inflammation?

DRG: Inflammation is the normal physiological response to tissue injury and infection. Most of the time our inflammatory responses are of an appropriate magnitude, they are quickly resolved and any damage to our tissues is successfully repaired. Inflammation is important because it drives the development of many important human diseases including rheumatoid arthritis, cardiovascular disease, inflammatory bowel disease and many others. Recent research suggests chronic inflammation may be an important driver of major mood disorders including depression.

My research is aimed at identifying endogenous pathways that are involved in regulating the magnitude and duration of inflammatory responses. Recently, we have been looking at the role of two independent cell signaling pathways in regulating the inflammatory response. One is centered on endocannabinoids a class of lipids that signals via a G protein coupled receptor (GPCR) called CB2 and the other pathway is centered on an unusual cytokine called Chemerin (TIG-2) whose effects are mediated by three different GPCRs ChemR23, CCRL2 and GPR1.

Macrophages in healthy and inflamed tissues play an essential role in the initiation and resolution of inflammation. One important aspect of macrophage biology in the context of inflammation resolution is phagocytosis of cellular debris and phagocytosis of neutrophils that have undergone apoptosis. Macrophage phagocytosis of apoptotic cells (efferocytosis) has a profound effect on inflammation resolution. Macrophage efferocytosis changes the profile of macrophage cytokine secretion towards a more anti-inflammatory / pro-resolution phenotype, which in turn will enhance inflammation resolution. Failure to clear apoptotic neutrophils from a site of inflammation can lead to failure of resolution and a substantially worse outcome caused by secondary necrosis.

The IncuCyte Live-Cell Analysis System enables detailed analysis of immune cell biology monitor changes in morphology and measure cell health, chemotactic migration and phagocytosis in real time. Automatically visualize the differentiation of immortalized THP-1 cells into M0 macrophages and qualitatively analyze the differentiation of primary monocytes into M1 and M2 macrophage populations

A powerful research tool

SN: How does the IncuCyte technology help you to achieve your research goals?

DRG: We have now been using the IncuCyte Live-Cell Analysis Systemto study several different aspects of macrophage cell biology in a wide range of different applications. We have found this real time live cell imaging system to be easy to use and the associated image analysis software makes this a very powerful research tool.

SN: How did you monitor cell behavior before you installed the IncuCyte? What does this technology enable you to do that you couldnt do before?

DRG: All the macrophage biology experiments that we have published using the IncuCyte platform could have been performed using other imaging modalities but I think that the big advantage of the IncuCyte live cell imaging platform lies in the ease of use and ease of analysis compared to other cell imaging methods (confocal microscopy, flow cytometry and Imagestream). What we particularly like about the IncuCyte system is the ability to develop protocols to study both generalized and cell type specific behavior. For instance we can follow proliferation or apoptosis of macrophages, and we can study macrophage specific cell behavior such as apoptosis or chemotaxis. Data analysis is greatly facilitated by user friendly software.

SN: What is next for your research?

DRG: I want to start using the IncuCyte system to do scratch wound migration assays where we look for macrophage secreted factors that play a role in wound repair processes. Hopefully we can scale up this cell-based assay to look for novel chemicals, peptides and proteins that will enhance tissue repair in the context of inflammation resolution.

The long-term goal of research in my laboratory is to turn high quality basic science into new treatments that enhance wound repair and help resolve chronic inflammation. Our ability to study both murine and human macrophages on the IncuCyte platform will be important in future translational research programs.

SN: Do you have any advice for other researchers who are considering using IncuCyte technology?

DRG: Take your time in setting up the assays before you pile in to testing lots of different mediators, drugs etc. Every cell type is different so one size fits all protocols are unlikely to work first time!

Read this article:
SelectScience Interview: Live Cell Analysis in Chronic Inflammation Research at the University of Oxford - SelectScience.net (blog)