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A Fresh Turn in Membrane Trafficking Research
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| Elucidation of the melanin transport mechanism | |||
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On November 15, 2004, newspapers featured articles with the headlines 'Melanin Transport Mechanism Elucidated, Applications to Prevent Gray Hair Expected' and 'Melanin Transport Mediator Identified, May Be Effective in Skin Whitening and Preventing Gray Hair!' The results of a cooperative study by Fukuda and Taruho Kuroda, a special postdoctoral researcher, hit the headlines. "I never dreamed we would get such an enormous response from readers, and I was astonished to get so many inquiries from workers at cosmetics manufacturers who read the articles," says Fukuda. Even now, he is receiving many requests for lectures and joint research. Melanin is a pigment that blackens the skin and hair. On the one hand it protects cells against ultraviolet light, which is harmful to organisms, but on the other hand it causes sunburn, chloasma, and freckles. Melanin is produced exclusively by melanocytes, a type of cell found inside the skin and at the roots of hairs (Figure 1). Formed around the melanocyte nucleus, melanin is stored in melanosomes and transported to the cell periphery along microtubules and actin filaments. Subsequently, the pigment is transferred to adjacent keratinocytes (skin cells) and hair matrix cells, thus making the skin and hair black. Although melanosomes have long been known to move in melanocytes because their movement is easy to observe using an optical microscope, the mechanism had remained unknown. Fukuda and others have elucidated the melanin transport mechanism at the molecular level. Being the object of public attention worldwide, Fukuda says with a laugh, "Few people in the pigment sector are aware of me. They may regard me as an upstart." He adds, "My work is backed by neurotransmitter release." Fukuda had been engaged in research into neurotransmitter release mechanisms at RIKEN's Brain Science Institute. How are neurotransmitter release and melanin transport linked together?
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| From neurotransmitters to melanin | |||
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Our body comprises 60 trillion cells. Each cell contains a variety of membrane-wrapped subcellular units known as organelles, which include the nucleus, Golgi apparatus, and endoplasmic reticulum. As an organelle, melanosome synthesizes and stores melanin. Membrane-wrapped substances are transported between organelles to mediate signal exchange. This is called membrane trafficking. The form of membrane trafficking that involves substance transport outside the plasma membrane is called secretion. Neurotransmitter release is a form of secretion. "A variety of organelles are present in a cell, each of which is involved in membrane trafficking. It is very important to life that membrane trafficking occurs in due order, and that signal exchange takes place accurately," says Fukuda. "To ensure systematic membrane trafficking, a traffic controller is necessary. I have been focusing on synaptotagmin, an important protein that controls neurotransmitter release. In the belief that some protein controls membrane trafficking outside the nervous system, I had been searching for a protein structurally similar to synaptotagmin. As a result, I discovered two protein families: the Slp (synaptotagmin-like protein) family and the Slp-like Slac2 (Slp homologue lacking C2 domains) family. This discovery led to my work on melanin transport." In humans, five members have been found in the Slp family, and three in the Slac2 family. Fukuda says, however, that at first he had absolutely no prospects for his work. "This was because no information was available on the function of Slp, except for its structural similarity to synaptotagmin. When Slp and Slac2 were found to bind to the Rab27A protein, my work accelerated dramatically." First, Fukuda attempted to identify proteins to which Slp and Slac2 bind. Both have a domain that is likely to bind to small GTP-binding proteins (G proteins). Hence, Fukuda took note of Rab, a group of small G proteins. Rab occurs in two forms: active and inactive. The active form is known to interact with specific proteins (effector molecules), and to play a key role in membrane trafficking. In humans, there are about 60 kinds of Rab that bind to different proteins, and that are responsible for the respective forms of membrane trafficking. I did not know which of the 60 kinds of Rab are the binding partner of Slp and Slac2. So I decided to try all cases. Although my approach may appear to be too exhaustive, I thought that steady one-by-one examination would lead to earlier completion of the work." As a result, Rab27A was identified as the binding partner at the end of 2001. "The gene for Rab27A was shown to be the causative gene for Griscelli syndrome in 2000. Out of the members of the Rab family, the Rab27A protein was drawing a lot of attention because it was the first to be proven to be associated with a hereditary disease." Griscelli syndrome is an autosomal recessive disorder characterized by pigment dilution of the skin and hair, immunodeficiency, and neurological disorder. Only about 50 cases have been reported to date worldwide. "Extensive examination to identify proteins that work at downstream of Rab27A and to clarify their functions would elucidate the mechanism behind skin and hair blackening. With the expectation of obtaining results that would lead to skin whitening and the prevention of gray hair, as well as to the treatment of Griscelli syndrome, I applied for RIKEN's year 2002 recruitment of initiative research scientists and began my current work to elucidate the pathology of Griscelli syndrome as the leader of the Fukuda Initiative Research Unit." |
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| Cooperation of two kinds of effector molecules | |||
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The first significant finding at the Fukuda Initiative Research Unit was that Rab27A, Slp2-a in the Slp family, and Slac2-a in the Slac2 family are present on melanosomes and mediate melanin transport (upper panel on cover page). In 2004, Fukuda and others succeeded in elucidating the mechanism behind melanin transport at the molecular level, as described at the beginning of this article (Figure 2). "Rab27A can be compared to a home delivery service tag" explains Fukuda. "Melanosome is a 'parcel' that bears the 'tag' for Rab27A. Melanosome is also bound with myosin Va, a kind of motor protein that serves as a 'truck,' and Slac2-a as a 'driver.' Hence, a total of three kinds of proteins are bound to the melanosome. The 'parcel' is trucked on a 'road' of actin filaments. When the 'truck' comes near the plasma membrane, the 'parcel' is passed to a 'deliverer' (Slp2-a). Subsequently, the 'deliverer' hands the 'parcel' to the plasma membrane." Although some kinds of Rab had been shown to be associated with more than one effector molecule, no one had any idea about how they utilize the effector molecules in distinct ways. Fukuda and others demonstrated for the first time in the world that a single kind of Rab27A sequentially utilizes the two kinds of effector molecules of Slac2-a and Slp2-a. This achievement is expected to find aesthetic applications in skin whitening and prevention of gray hair. "The currently available skin whiteners are mostly inhibitors of melanin synthesis. Our achievement demonstrates the potential of melanin transport for a new target of skin whitening. We have been offered joint research with a cosmetics manufacturer." However, problems remain to be solved before the mechanism behind melanin transport is elucidated. "The point to be clarified resides in how the melanosomes are transported along the microtubules, and how melanin is transferred to keratinocytes and hair matrix cells. I think it is highly likely that a kind of Rab other than Rab27A works in this process." Fukuda says, "Rab27A, Slp, and Slac2 are now attracting great attention from many researchers in the world. Our work has demonstrated that these proteins are associated not only with some diseases and melanin transport, but also with the control of the endocrine secretion, exocrine secretion, and secretion of immune granules, thus creating a worldwide sensation among the researchers in relevant fields. Competition will heat up." Then, what strategy are they adopting to advance their work at the Fukuda Initiative Research Unit? "Unlike many other researchers focusing on the function of Rab27A, we are working from the viewpoint of effector molecules. We are constructing a system that would enable comprehensive search for effector molecules for the 60 kinds of Rab. Although I cannot yet give any details about this project, the system would revolutionize research into membrane trafficking."
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| Elucidation of membrane trafficking in higher organisms | |||
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Although Rab is found in all eukaryotic organisms, from yeast to mammals, it varies in number among different species. For example, humans have about 60 kinds of Rab, whereas budding yeast has 11 kinds only. Fukuda explains the reason for this difference, "Humans seem to require special modes of membrane trafficking for survival as multicellular organisms. The first to eleventh Rab are conserved between yeast and humans, and are considered to mediate the minimum essential membrane trafficking processes for the survival of all cells. The twelfth Rab and beyond, particularly the second half of them, are considered to be responsible for key functions for intercellular communication and survival as higher organisms. We are arranging a project to extensively examine the twelfth Rab and beyond, which have not yet been investigated in full. The project will certainly provide a new insight into membrane trafficking." Finally, Fukuda talked about the future prospects for his work, "Membrane trafficking also plays a key role in fertilization and embryogenesis. I want to open the frontiers of research into biological phenomena from the viewpoint of membrane trafficking." 
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The human body comprises a great many cells, each of which contains many subcellular units known as organelles. Signals are exchanged frequently between cells and between organelles through membrane trafficking in which membrane-wrapped substances are transported. However, much remains unknown about this process. At the Fukuda Initiative Research Unit, researchers are conducting investigations focusing on the transport of the melanin pigment and on the secretion out of the various forms of membrane trafficking. Recently, the Unit drew attention by elucidating the mechanism of melanin transport at the molecular level for the first time in the world. Mitsunori Fukuda, the initiative research scientist who leads the Unit, says, "Rab27A and the Slp and Slac2 families are proteins responsible for melanin transport and are drawing the attention of researchers in the world as being associated with various secretory phenomena and Griscelli syndrome, a hereditary disease characterized by pigment dilution. Competition will heat up for remarkable achievements with these proteins." This article reports on this revolutionary research into membrane trafficking.
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Discovery of the New "RNA Continent" Disproves Common Knowledge on Genomes!
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| Full-length cDNAs to compete with Europe and the Unites States | |||
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In 1995, Dr. Hayashizaki was appointed to Genome Project Director at RIKEN. "At that time, the United States declared that it would finish sequencing the human genome by the end of 2003. Venture companies supported by giant pharmaceutical firms in Europe or the United States focused attention on fragments of cDNAs (complementary DNAs) and tried to sequence every single piece. Under the circumstances in which Japan lagged behind Europe and the United States in the development of Genome Science, we thought on what to do and started a full-length mouse cDNA project." To begin with, what is cDNA? There are four different bases in a DNA molecule: adenine (A), thymine (T), guanine (G), and cytosine (C), and the sequence of these bases holds genetic information (Figure 1). In DNA, two strands form a double stranded structure where A and G are complementarily connected to T and C, respectively. Genes are comprised of both transcribable and untranscribable DNA sequences, where the transcribable DNA sequences are to produce proteins. During the process of expression, the double stranded structure comes unbound and the RNAs complementary to the base sequence of the original strand are formed. Thus the gene information is read out (transcription). Then sections of the RNAs are separated (splicing) and some segments, called exons, are connected to form mRNAs. Proteins are produced based on the information stored in mRNAs (translation). The mRNA information can be artificially copied to DNA sequences that are chemically stable and easily replicable and amplified in hosts such as E. coli. These DNAs are referred to as cDNAs. "The United States played a central role in sequencing the human genome that is composed of about three billion base pairs, and it has been considered that only 2% of them are actually transcribed. Sequencing the genome base sequences themselves, however, does not supply us with the necessary information as to which sections are transcribed to RNAs, and in what order the RNAs are connected to form mature mRNAs. Thus, we have no alternative but to look into mRNAs/cDNAs to obtain this information." Note that in 1995 we were still unsuccessful in establishing a technique to copy a full-length mRNA sequence to a cDNA from one end to the other. What had been done was only to form segmented cDNAs with incomplete information. Therefore, his team began developing a unique technology to create a full-length cDNA, where the complete information of the mRNA is copied. It is only with the full-length cDNA that all the necessary information to produce proteins can be analyzed, and thereby the proteins can be manufactured and reproduced. "We set our target to compile a mouse genome encyclopedia." All the full-length mouse cDNAs are obtained and their base sequences are decoded before assembling a database, where the function of each sequence is annotated. But why mouse cDNAs rather than human? "To obtain full-length cDNAs, we need mRNAs, and to gather all mRNAs, we need to gather mRNAs in various tissue cells from each development stage that begins with a fertilized egg. Thus we concluded that mouse mRNAs are better because of the limited use of human mRNAs." Mice have almost all the genes and diseases that are observed in humans. The full-length mouse cDNA database is an important research infrastructure for the life sciences and medical science. 
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| Mouse genome encyclopedia | |||
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Dr. Hayashizaki and his team have established a unique technology to make a full-length cDNAs. They have also built up a unique fast sequencing system that is used to sequence their nucleotide sequences, and released the full-length mouse cDNAs database to the public. They have played host to the international organization "FANTOM," and pushed ahead with its high throughput analysis. Thus in 2002, they finally completed the "Mouse Genome Encyclopedia" that includes the data of about 60,000 full-length cDNAs. "The nucleotide sequencing technology is advancing rapidly. The full-length cDNA technology, however, is still the strong point of the Japanese life sciences." For example, in Japan, RIKEN is taking the leading role in the "Protein 3000" project with the goal of determining the structures and functions of a large number of important proteins. This world-leading project would not be possible without the full-length cDNA technology used for protein synthesis, and the accumulated data. Dr. Hayashizaki and his team have also developed another innovative and unique technology that can dramatically enhance the research efficiency based on the full-length cDNA technology. Focusing attention on the stability of cDNA as a chemical substance, they developed what they call DNABook® where the full-length cDNAs they obtained are printed. In the past, the full-length cDNAs were integrated in E. coli and frozen before they were stored or transported. This method was troublesome, time-consuming, and costly. In contrast, the DNABook® is stored or transported at room temperature. This book is very handy for researchers because they can take the book from the bookshelf before starting performance analysis. "This technology can be extended in the near future so that samples of cells or viruses can be stored in printed form." |
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| Understanding the genome network | |||
Dr. Hayashizaki and his team have launched the "Genome Network Project" based on the full-length cDNA database. "We can understand how each gene functions and that mutation of a gene leads to a disease. However, why does the mutation cause the disease? We do not know the mechanism." This is because a gene does not function in isolation, but as a member of a network consisting of many genes and proteins (Figure 2). For example, a protein produced from causative gene A can induce the expressions of genes B, D, and E. Then, the protein that is produced from B, in turn, can affect genes C, G, and F, finally causing disease. The Genome Network Project aims at throwing light on the functional network of these genes and proteins. This approach will lead us to the first understanding of the mechanism of disease at the molecular level, which will result in the development of drugs with low possibility of side effects. |
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| Impact of the RNA continent | |||
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From here, let us go back to the new findings that have revolutionised the common knowledge on genomes. In 2002, the database consisting of about 60,000 full-length cDNAs was released to the public, where many non-coding RNAs (ncRNAs) were found. "In the past, only 100 ncRNAs were known to the public, but many more ncRNAs, or what we call an "RNA continent" were found in the database. This discovery brought sudden progress in our research program." The database released this time includes 23,000 or more ncRNAs; the number accounts for 53% of the total number of RNAs obtained. Furthermore, the rate tends to increase as the research advances. "This is my estimation, but if the RNAs that have not yet been obtained are included, the number of all the RNAs that are transcribed from genomes will add up to about one million. Among them, the number of RNAs that can produce proteins will account for from one-tenth to one-eighth, and most of them are ncRNAs." Then, what do they do in tissue cells? In 2002, the US science magazine Science featured the discovery of RNA interference (RNAi) as the lead story of the year. RNA interference is an innovative technology that can inhibit the function of specific genes. In other words, when double strand RNAs including base sequences complementary to those of the mRNAs produced by the gene we want to inhibit are injected into cells, the double strand RNAs link together with the target mRNAs and can inhibit the synthesis of specific proteins. "RNA interference is a man-made technology. However, nature has already used the same mechanism within a cell." Among the ncRNAs in the full-length cDNAs database, a large number of ncRNAs having sequences complementary to those of RNAs that produce proteins have been found. "It is amazing, but the information of the counter strands was also read out, which form a strand pair with the DNA strands that carry the information on proteins we pay attention to. We do not know how the ncRNAs function, but they may inhibit the synthesis of proteins with the same mechanism as observed in the RNA interference. At present it is clarified that, in addition to RNA interference, the ncRNAs can affect DNA in the nucleus, and through the proteins in the nucleus, they can inhibit or promote or cut out a part of a DNA molecule to form a recombined genome. It seems that a considerable number of ncRNAs are "functional RNAs" that have functions of this kind. Furthermore, a new fact that disproves the common knowledge on genomes was found. It has been anticipated that about 2% of RNA information is read out. The fact, however, is that it is about 70%. "Most genomes have been considered to be junk, but it is not true." It was also proved that genome information is read out in various manners. It is read out from various points in the gene region. A genome is cut out in a varied splicing manner to produce many kinds of mRNAs. Some RNAs were found that transversely read out the information of two separate genes far apart from each other. "Thus, it becomes meaningless to discuss the number of genes. For example, the number of fly genes is somewhere between 10,000 and 20,000 whereas the number of human genes is about 22,000; there is no significant difference. Then, why do human beings have advanced functions? This is because human genomes are read out in a varying manner, which contributes to form various kinds of proteins from a single gene region. Furthermore, it is considered that RNAs take the leading role in sophisticatedly controlling the functions of DNAs. To create the bodies of complex higher organisms and to allow them to fulfill their functions, it is necessary for the functions of DNAs to be controlled extremely precisely. The newly discovered RNA continent is an undeveloped continent. In the future, it is necessary to clarify RNA functions in more detail and their roles in the genome network. This is very important for overcoming diseases because many ncRNAs with complementary sequences to those of the genes which cause diseases are discovered. For example, some cells may maintain a healthy condition because those ncRNAs can properly affect causative genes to inhibit the synthesis of proteins that would be produced by the causative genes. In fact, some examples show that some diseases are due to the mutation of ncRNAs. "Beginning with a fertilized egg, life involves the three stages of birth, growth and aging. In all the cells at every stage, how is genome information used to form RNAs or how do those RNAs build a functional network with DNAs and proteins? To study the issues chronologically, we need to launch a research project into the 'dynamic system biology' at some time in the future. (Lower picture on the cover page: RIKEN developed 3D-viewer displaying the expression of genes. The 3D-viewer can display four parameters: expression stage, tissue, locus, and expression level.) However, note that we need a supercomputer that is capable of dealing with a vast amount of data." Project Director Dr. Hayashizaki is setting his sights on new developments in life sciences over the horizon of the vast RNA continent.
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Genetic information stored in DNA is read out to produce proteins, and the proteins in turn fulfill their functions to support life phenomena. In the past, it was considered that RNAs read out about 2% of all genome information or all genetic information, and that they were only a means of communication between DNA and proteins. In September 2005, however, Dr. Yoshihide Hayashizaki, Project Director at the RIKEN Yokohama Institute et al., published their findings in the September 2 issue of the US science magazine "Science." "We have known that most RNAs do not produce proteins. Instead, they control DNA functions. Every life scientist should clearly understand this new fact before they proceed to further studies," says Dr. Yoshihide Hayashizaki, Project Director, during an impromptu interview.