How do cells form organs? That is, how does each cell within an organ assume the correct shape and form the right contacts with its neighbors?
Those are the questions that interest Max Heiman, a postdoc in Shai Shaham's lab at the Rockefeller University in New York, who earlier this year showed how two proteins shape the neurons and glia of the C. elegans sensory organ. The two proteins - DEX-1 and DYF-7 - anchor the neuronal dendrites at the nose of the worm while the cell body migrates away, eventually forming an axon that projects into the worm brain. Both proteins are secreted, and seem to form an extracellular matrix that prevents the dendrite from being dragged away with the cell body, helping the neurons form their correct, elongated shape. Heiman has now extended this work by asking whether other worm neurons similarly depend on DEX-1 and DYF-7. The answer is no - but he's identified mutants that specifically affect other groups of neurons instead. Different neurons therefore seem to use distinct machinery to form their correct contacts and shape. Especially intriguing is Heiman's idea that components of the extracellular matrix can play specific roles in shaping cells - it's rather like guy ropes giving shape to a tent in addition to the internal framework provided by tent poles.
Image of a worm sensory neuron from Evans et al, J Cell Biol, 2006.
On Tuesday morning, I attended the minisymposium on the Functional Organization of Plasma Membranes, organized by Matthew Rasband (Baylor College of Medicine, Houston) and the absent-due-to-illness Benedicte Dargent (Universitee de la Mediterranee).
Most of the talks in the session focused on the organization of integral membrane proteins into specific domains by the spectrin-ankyrin membrane skeleton. Spectrin forms an interconnected meshwork on the inner surface of plasma membranes and is linked to specific transmembrane proteins by ankyrin adaptors. Late last year, Gai Ayalon, from Vann Bennett's lab at Duke University, showed that ankyrin B organizes dystrophin and dystroglycan at specialized skeletal muscle membrane structures called costameres. Dystrophin and dystroglycan are vital for linking the muscle fiber cytoskeleton to the surrounding extracellular matrix, and neither protein is properly localized in the absence of ankyrin B. In addition, ankyrin B interacts with dynactin-4, a subunit of the dynactin complex that links the motor protein dynein to cell membranes. On Tuesday, Ayalon presented studies that further characterized the association between ankyrin B and dynactin-4, and investigated the role that this interaction plays in organizing proteins at the costamere.
Matthew Rasband then discussed the role of spectrin and ankyrin in organizing a specialized membrane domain in neurons called the axon initial segment (AIS). This domain, which contains specific cell adhesion molecules and voltage gated ion channels in addition to spectrin and ankyrin, is the axonal gatekeeper - it makes a decision on whether or not to fire an action potential in response to the cumulative signal it receives from the neuron's dendrites. Rasband presented data from his group's recent paper, demonstrating that neuronal injury (e.g. by ischaemia) causes the calcium-dependent protease calpain to cleave spectrin and ankyrin G, resulting in AIS disassembly and a loss of neuronal polarity. This loss was irreversible, but calpain inhibitors blocked AIS disruption following injury, which could be a useful approach for treating stroke patients.
Meanwhile, Bettina Winckler, from the University of Virginia, is interested in how a cell adhesion molecule called Neurofascin is specifically localized to the AIS, and nowhere else along the axon. Voltage-gated sodium channels are specifically targeted to the AIS through a combination of retention by binding to ankyrin, and endocytic removal from other membrane domains (see Fache et al, 2004). Winckler showed that a similar mechanism is used to accumulate Neurofascin at the AIS, although with a twist: Neurofascin doesn't bind directly to the clathrin endocytic machinery, but NGF signaling promotes its association with a protein that links the cell adhesion molecule to clathrin cargo adaptors. NGF therefore enhances Neurofascin's endocytosis from sites along the axon and dendrites, promoting its unique accumulation at the AIS, where it stably binds ankyrin and isn't internalized.
The ability of spectrin and ankyrin to capture proteins at specialized membrane domains is one of the network's main functions, along with providing shape and support to the plasma membrane as a whole, particularly in red blood cells. Both of these functions require a high amount of spectrin and ankyrin that interact with binding partners in a roughly 1:1 stoichiometry. But G. Harper Mazock, from Ron Dubreuil's lab at the University of Illinois, Chicago, presented evidence that there's a third, essential function for spectrin that only requires small amounts of the protein. This was based on their observations that flies can tolerate a reduction in beta-spectrin levels within their nervous system of more than 80% (through tissue-specific RNAi). Although these flies have a reduced life span, complete removal of beta-spectrin from the nervous system is embryonic lethal. This suggests that there's a "low-abundance tier" of spectrin function that, based on previous reports, may correspond to a spectrin population involved in membrane trafficking. It seems that spectrin and ankyrin act to organize membrane domains in many different ways.
Sunday saw the first minisymposia at this year's ASCB meeting. I decided to attend the session on Intracellular Trafficking, organized by Joachim Seemann (UT Southwestern, Dallas) and Elizabeth Miller (Columbia University, New York). There were some really great talks that touched on many different aspects of transport - I particularly enjoyed the presentation from Miller herself, who described a new mechanism by which cargo capture is linked to vesicle production.
COPII vesicles transport cargo from the ER to the Golgi, and get their name from the assembly of proteins that coat their outer surface. Assembly of these coat proteins is regulated by the small GTPase Sar1, whose cycle between GTP and GDP-bound states is controlled in turn by other components of the COPII coat. Coat assembly is initiated by Sar1-GTP, while Sar1-GDP triggers COPII disassembly. The GTPase cycle takes about 10 seconds - is this enough time to form a productive vesicle packed with cargo for delivery to the Golgi?
Sec24, meanwhile, is the COPII cargo adaptor, which recruits cargo into vesicles forming at the ER through at least 3 different cargo binding sites. While looking for additional binding sites, Miller's group discovered a Sec24 yeast mutant with an unusual phenotype - while it could form COPII vesicles just as efficiently in the presence of the non-hydrolyzable GTP analog GMP-PNP, vesicle production was reduced in the presence of GTP itself. Miller's team found that the Sec24 mutation abolishes the protein's interaction with a COPII scaffold protein called Sec16. And while, in wild type cells, Sec16 can delay Sar1's GTP cycle, it can't maintain active Sar1 in the Sec24 mutant. Miller thinks that Sec24 couples cargo capture to a Sec16-mediated inhibition of coat disassembly, allowing enough time for a proper vesicle to form. If cargo is absent (or the Sec24-Sec16 interaction is prevented) the COPII coat will disassemble from the ER membrane before a useless, empty, vesicle forms.
The next talk, by Rodney Infante from Michael Brown and Joseph Goldstein's lab at UT Southwestern, dealt with transport of a very different kind. Cholesterol is unpackaged from LDL particles by acid lipases in the lysosomes. The lipid must then be moved to the ER, where it is processed for storage in lipid droplets. But cholesterol has to be kept in a hydrophobic environment throughout these steps, to prevent it from aggregating and damaging the cell. This doesn't happen in Niemann-Pick type C patients, resulting in cholesterol buildup, neurological problems, and patient mortality. Infante - who was awarded the Norton B. Gilula award, sponsored by the Rockefeller University Press - gave a great talk describing the function of the two lysosomal proteins mutated in Niemann-Pick type C, NPC1 and NPC2. In a paper earlier this year, the group showed that the two proteins bind cholesterol in different ways, and might participate in a "hydrophobic handoff", in which the soluble NPC2 protein rapidly binds cholesterol liberated from LDL particles before passing it on to the transmembrane protein NPC1 in the correct orientation for its insertion into the lysosomal membrane. Infante now described the group's search for point mutations that disrupt this cholesterol transfer step and which can't rescue cholesterol storage defects of cells lacking wild type NPC1. It's really exciting work that explains a serious human disease. Intriguingly though, no one seems to know what happens after cholesterol has been transferred to the lysosomal membrane and how it goes from there to the ER...
The final talk in the session was from Jen-Hsuan Wei, a student from Joachim Seemann's lab, who described in the JCB earlier this year how the mitotic spindle mediates inheritance of the Golgi apparatus' ribbon structure. Wei gave an overview of this work (you can read my summary here) before presenting some preliminary data suggesting that the Golgi reciprocates by promoting the assembly of the mitotic spindle - the picture on the left shows how mitotic Golgi fragments (green) cluster at the poles of the mitotic spindle (red). This intriguing talk, along with other presentations in the minisymposium that discussed cilia biogenesis and phagocytic clearance of apoptotic cells, showed how intracellular trafficking touches on many different aspects of cell biology.
We're in San Diego for the 49th annual meeting of the American Society for Cell Biology. Although there were some small sessions earlier in the day, the conference really kicked off on Saturday evening with a keynote address from Rudolf Jaenisch. The huge auditorium was packed with cell biologists, eager to hear Jaenisch discuss his latest work on induced pluripotent stem cells, but it was interesting to hear Jaenisch begin his talk with a description of his early scientific career - including the creation, with Beatrice Mintz, of the first transgenic mouse. It was fascinating to hear Jaenisch discuss his struggles with the technological limitations of the time - how do you know that a mouse is transgenic when southern blots haven't been invented yet?
Of course, Jaenisch eventually ended up working on epigenetics and nuclear reprogramming - a field that has been revolutionized in recent years by Shinya Yamanaka's demonstration that the expression of just 4 genes was sufficient to revert adult cells to an embryonic stem cell-like state. Jaenisch is interested in how the reprogramming event occurs: can all cells be reprogrammed, or just an "elite" subset of them? Is the process deterministic (every cell that can reprogram does so in the same way at the same time), or is it stochastic (every cell reprograms with a random latency period)? Jaenisch presented his data - published this week in Nature - showing that reprogramming is a stochastic process and that every cell is capable of it. The chance of reprogramming depends on the number of cell divisions. Speeding up proliferation by removing p53 increased the rate of iPS cell production (which is why, as several papers showed earlier this year, p53 inhibition appears to enhance reprogramming efficiency). In contrast, overexpressing the transcription factor Nanog genuinely enhances the efficiency of iPS cell production, allowing reprogramming to occur over fewer cell divisions.
Jaenisch ended his presentation with a discussion of the future prospects and challenges for iPS cell technology, both in the lab and the clinic. iPS cells derived from patients with serious conditions like Parkinson's disease, could be redifferentiated in vitro into diseased tissues, which could then be used as model systems for basic research and drug screening. However, 3D culture systems will need to be developed that will allow the differentiated iPS cells to accurately recreate diseased tissue - Jaenisch suggested that, controversial as it may be, human-mouse chimeric animals generated with human-derived iPS cells might prove to be the best tool for these purposes. Meanwhile, Jaenisch's group demonstrated in 2007 that iPS cell technology can be applied in the clinic, when they treated mice with sickle cell anemia by transplanting iPS cell-derived hematopoietic progenitors. But several barriers remain before the approach can be used on human patients - including learning how to produce iPS cells on a large scale and how best to deliver them.
While it's important that the public doesn't develop unrealistic expectations from these exciting developments, it's also vital for society to appreciate the work that scientists are doing. That was the message from NIH director Francis Collins, who made an unscheduled appearance on stage immediately after Jaenisch's talk. Collins began by assuring the audience that, contrary to the complaints made by some upon his appointment earlier this year, he wasn't only interested in "Big Science". He recognizes the importance of basic research initiated by individuals, and cited this year's Nobel Prize winners as examples. He then discussed how the NIH spends its budget, particularly the $10 billion it received as part of the stimulus package, and encouraged biologists to report how the extra funds had helped them continue important research in the current economic climate. Indeed, the ASCB has set up a special website for scientists to tell their stories of what the funding has done for them. Collins highlighted other ways in which we can educate the public about the importance of basic research, including inviting politicians to your laboratory, or taking part in National Lab Day. Of course, not all of us can do scientific outreach by appearing on the Colbert Report, like Collins did in October!
biowrites content is distributed under the terms of an Attribution-Noncommercial-Share Alike-No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution-Noncommercial-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).