Чудаков Дмитрий Михайлович

Личная информация

Чудаков Д. М. окончил Московский Государственный Университет им. М. В. Ломоносова. Работает в  ИБХ РАН с 2000 года. В 2003 году, под руководством д. б. н. К.А. Лукьянова, защитил кандидатскую диссертацию по теме «Фотоконверсия окрашенных белков из коралловых полипов». C 2008 года возглавляет группу Флуоресцентных инструментов для иммунологии и нейробиологии ИБХ РАН. В 2011 году защитил докторскую диссертацию по теме "Генетически кодируемые флуоресцентные инструменты для исследования живых систем".

 

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
1995–2000 Россия, Москва МГУ им. Ломоносова

Премии и заслуги

1. Медаль Российской академии наук с премией для молодых ученых РАН, 2005 г.

2. Медаль Европейской Академии наук, 2006 г.

3. Лауреат программы "Выдающиеся ученые. Кандидаты и доктора наук РАН", 2006-2007, 2008-2009 гг.

4. Лауреат грантов Президента Российской Федерации молодым российским ученым, 2006-2007, 2008-2009 гг.

5. Диплом I степени МАИК за лучшую публикацию в журнале «Биоорганическая химия» за 2007 год.

6. Премия  конкурса МАИК «Наука/Интерпериодика» на лучшую публикацию по биологическим наукам за 2008 год.

7. Диплом за лучшую публикацию в журналах РАН за 2008 год.

8. Диплом II степени МАИК за лучшую публикацию в журнале «Биоорганическая химия» за 2010 год.

9. Диплом за наиболее цитируемую за 2011 год публикацию в журналах группы BJ Cell.

Основные научные результаты

Чудаков Д. М. расшифровал механизм обратимой фотоактивации флуоресценции, подразумевающий цис-транс изомеризацию хромофора, и получил целый ряд так называемых фотоактивируемых белков с высоким контрастом активации. Флуоресцентные характеристики таких белков могут обратимо либо необратимо изменяться в ответ на облучение светом определенной длины волны, что предоставляет уникальные возможности для прицельной маркировки и слежения за живыми объектами, а также для технологий флуоресцентной микроскопии сверх-высокого разрешения.

Чудаков Д. М. разработал широкую палитру ярких мономерных флуоресцентных белков, флуоресцирующих в диапазоне всего видимого спектра и позволяющих проводить многоцветное мечение молекул в живых клетках: TagBFP, TagCFP, TagGFP, TagYFP, TagRFP, а также дальне-красные белки mKate и mKate2. Белок TagRFP является самым ярким из разработанных мономерных красных флуоресцентных белков, существенно опережая немногие имеющиеся аналоги, и хорошо зарекомендовал себя в химерных конструкциях с различными исследуемыми белками. Белок mKate2 является абсолютным лидером по яркости среди мономерных дальне-красных флуоресцентных белков.

Чудаковым Д. М. разработаны яркие быстро созревающие белки: TurboGFP, TurboYFP, TurboRFP, TurboFP602, а также яркий дальне-красный белок Катюша (Katushka, TurboFP635). Уникальное для флуоресцентных белков сочетание высокой яркости и длинноволнового излучения позволяет эффективно детектировать сигнал Катюши в живых тканях, проницаемость которых повышена в дальне-красной области спектра. Катюша существенно повышает чувствительность технологий прижизненной визуализации клеток и тканей в интактных трансгенных организмах, и в перспективе может найти свое применение в практической медицине.

Чудаковым Д. М. также получен ряд высоко-контрастных генетически кодируемых сенсоров на основе флуоресцентных белков, в том числе сенсоров концентрации кальция, сенсоров программируемой клеточной гибели.

С участием Чудакова Д. М. получен первый и единственный на сегодняшний день генетически кодируемый фотосенсибилизатор KillerRed, ведутся исследования его биохимии и разработка прикладных технологий.

Разработанные Чудаковым Д. М. флуоресцентные инструменты широко используются в научных лабораториях и на фармацевтических компаниях. Чудаков Д. М. является автором более 50 статей в рецензируемых журналах. Индивидуальный индекс цитирования: 1900. Индекс Хирша: 20.

Чудаков Д. М. принимал участие более чем в 25 международных конференциях в 10 странах, в большинстве случаев в качестве докладчика.

Чудаков Д. М. является соавтором ряда патентов и  международных патентных заявок по флуоресцентным белкам.

Под руководством Чудакова Д.М. были подготовлены и успешно защищены кандидатские диссертации по теме разработке новых инструментов на основе флуоресцентных белков:

Суслова Екатерина Андреевна, «Высококонтрастные генетически кодируемые сенсоры на основе зеленого флуоресцентного белка», 2008 год, по специальности  «Молекулярная биология».

Щербо Дмитрий Сергеевич, «Дальнекрасные флуоресцентные белки», 2010 год, по специальности  «Молекулярная биология».

Избранные публикации

  1. Mamedov I.Z., Britanova O.V., Bolotin D.A., Chkalina A.V., Staroverov D.B., Zvyagin I.V., Kotlobay A.A., Turchaninova M.A., Fedorenko D.A., Novik A.A., Sharonov G.V., Lukyanov S., Chudakov D.M., Lebedev Y.B. (2011). Quantitative tracking of T cell clones after haematopoietic stem cell transplantation. EMBO Mol Med 3 (4), 201–7 [+]

    Autologous haematopoietic stem cell transplantation is highly efficient for the treatment of systemic autoimmune diseases, but its consequences for the immune system remain poorly understood. Here, we describe an optimized RNA-based technology for unbiased amplification of T cell receptor beta-chain libraries and use it to perform the first detailed, quantitative tracking of T cell clones during 10 months after transplantation. We show that multiple clones survive the procedure, contribute to the immune response to activated infections, and form a new skewed and stable T cell receptor repertoire.

  2. Luker K.E., Mihalko L.A., Schmidt B.T., Lewin S.A., Ray P., Shcherbo D., Chudakov D.M., Luker G.D. (2011). In vivo imaging of ligand receptor binding with Gaussia luciferase complementation. Nat. Med. 18 (1), 172–7 [+]

    Studies of ligand-receptor binding and the development of receptor antagonists would benefit greatly from imaging techniques that translate directly from cell-based assays to living animals. We used Gaussia luciferase protein fragment complementation to quantify the binding of chemokine (C-X-C motif) ligand 12 (CXCL12) to chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. Studies established that small-molecule inhibitors of CXCR4 or CXCR7 specifically blocked CXCL12 binding in cell-based assays and revealed differences in kinetics of inhibiting chemokine binding to each receptor. Bioluminescence imaging showed CXCL12-CXCR7 binding in primary and metastatic tumors in a mouse model of breast cancer. We used this imaging technique to quantify drug-mediated inhibition of CXCL12-CXCR4 binding in living mice. We expect this imaging technology to advance research in areas such as ligand-receptor interactions and the development of new therapeutic agents in cell-based assays and small animals.

  3. Shcherbo D., Shemiakina I.I., Ryabova A.V., Luker K.E., Schmidt B.T., Souslova E.A., Gorodnicheva T.V., Strukova L., Shidlovskiy K.M., Britanova O.V., Zaraisky A.G., Lukyanov K.A., Loschenov V.B., Luker G.D., Chudakov D.M. (2010). Near-infrared fluorescent proteins. Nat. Methods 7 (10), 827–9 [+]

    Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

  4. Zvyagin I.V., Mamedov I.Z., Britanova O.V., Staroverov D.B., Nasonov E.L., Bochkova A.G., Chkalina A.V., Kotlobay A.A., Korostin D.O., Rebrikov D.V., Lukyanov S., Lebedev Y.B., Chudakov D.M. (2010). Contribution of functional KIR3DL1 to ankylosing spondylitis. Cellular & molecular immunology , [+]

    Increasing evidence points to a role for killer immunoglobulin-like receptors (KIRs) in the development of autoimmune diseases. In particular, a positive association of KIR3DS1 (activating receptor) and a negative association of KIR3DL1 (inhibitory receptor) alleles with ankylosing spondylitis (AS) have been reported by several groups. However, none of the studies analyzed these associations in the context of functionality of polymorphic KIR3DL1. To better understand how the KIR3DL1/3DS1 genes determine susceptibility to AS, we analyzed the frequencies of alleles and genotypes encoding functional (KIR3DL1*F) and non-functional (KIR3DL1*004) receptors. We genotyped 83 AS patients and 107 human leukocyte antigen (HLA)-B27-positive healthy controls from the Russian Caucasian population using a two-stage sequence-specific primer PCR, which distinguishes KIR3DS1, KIR3DL1*F and KIR3DL1*004 alleles. For the patients carrying two functional KIR3DL1 alleles, those alleles were additionally genotyped to identify KIR3DL1*005 and KIR3DL1*007 alleles, which are functional but are expressed at low levels. KIR3DL1 was negatively associated with AS at the expense of KIR3DL1*F but not of KIR3DL1*004. This finding indicates that the inhibitory KIR3DL1 receptor protects against the development of AS and is not simply a passive counterpart of the segregating KIR3DS1 allele encoding the activating receptor. However, analysis of genotype frequencies indicates that the presence of KIR3DS1 is a more important factor for AS susceptibility than the absence of KIR3DL1*F. The activation of either natural killer (NK) or T cells via the KIR3DS1 receptor can be one of the critical events in AS development, while the presence of the functional KIR3DL1 receptor has a protective effect. Nevertheless, even individuals with a genotype that carried two inhibitory KIR3DL1 alleles expressed at high levels could develop AS.Cellular & Molecular Immunology advance online publication, 6 September 2010; doi:10.1038/cmi.2010.42.

  5. Chudakov D.M., Matz M.V., Lukyanov S., Lukyanov K.A. (2010). Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90 (3), 1103–63 [+]

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

  6. Bogdanov A.M., Bogdanova E.A., Chudakov D.M., Gorodnicheva T.V., Lukyanov S., Lukyanov K.A. (2009). Cell culture medium affects GFP photostability: a solution. Nat. Methods 6 (12), 859–60
  7. Pletnev S., Gurskaya N.G., Pletneva N.V., Lukyanov K.A., Chudakov D.M., Martynov V.I., Popov V.O., Kovalchuk M.V., Wlodawer A., Dauter Z., Pletnev V. (2009). Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J. Biol. Chem. 284 (46), 32028–39 [+]

    KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

  8. Mamedov I.Z., Britanova O.V., Chkalina A.V., Staroverov D.B., Amosova A.L., Mishin A.S., Kurnikova M.A., Zvyagin I.V., Mutovina Z.Y., Gordeev A.V., Khaidukov S.V., Sharonov G.V., Shagin D.A., Chudakov D.M., Lebedev Y.B. (2009). Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients. Autoimmunity 42 (6), 525–36 [+]

    Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

  9. Serebrovskaya E.O., Edelweiss E.F., Stremovskiy O.A., Lukyanov K.A., Chudakov D.M., Deyev S.M. (2009). Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein. Proc. Natl. Acad. Sci. U.S.A. 106 (23), 9221–5 [+]

    Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185(HER-2-ECD) antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185(HER-2-ECD)-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.

  10. Bogdanov A.M., Mishin A.S., Yampolsky I.V., Belousov V.V., Chudakov D.M., Subach F.V., Verkhusha V.V., Lukyanov S., Lukyanov K.A. (2009). Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol.  (5), 459–461 [+]

    Proteins of the green fluorescent protein (GFP) family are well known owing to their unique biochemistry and extensive use as in vivo markers. We discovered that GFPs of diverse origins can act as light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, via green-to-red GFP photoconversion, this process can be observed in living cells without additional treatment.

  11. Shcherbo D., Murphy C.S., Ermakova G.V., Solovieva E.A., Chepurnykh T.V., Shcheglov A.S., Verkhusha V.V., Pletnev V.Z., Hazelwood K.L., Roche P.M., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M. (2009). Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418 (3), 567–74 [+]

    Разработан яркий, мономерный, фотостабильный, pH-стабильный, дальне-красный флуоресцентый белок mKate2. Белок mKate2 хорошо показал себя в качестве метки во фьюзах с рядом белков, как в культуре клеток, так и в трансгенных лягушках Xenopus laevis (совместно с лабораторией Молекулярных основ эмбриогенеза ИБХ РАН).

  12. Serebrovskaia E.O., Stremovskiĭ O.A., Chudakov D.M., Lukianov K.A., Deev S.M. (2009). [Genetically encoded photoimmunosensitizer]. Bioorg. Khim. 37 (1), 137–44 [+]

    Photosensitizer-antibody conjugates are successfully used for targeted elimination of cancer cells bearing specific membrane markers. This method is known as photoimmunotherapy. However, chemical conjugation of photosensitizer and antibody poses a number of complications such as low reproducibility, aggregation and unconjugated photosensitizer impurities. Here we report a fully genetically encoded photoimmunosensitizer, consisting of an anti-HER2/neu miniantibody 4D5scFv and a phototoxic fluorescent protein KillerRed. Both domains in this photoimmunosensitizer retained their functional qualities - high affinity for HER2/neu antigen and phototoxicity respectively. 4D5scFv-KillerRed fusion protein showed high specificity for HER2/neu-over-expressing cells and effectively lowered their viability upon illumination.

  13. Shcherbo D., Merzlyak E.M., Chepurnykh T.V., Fradkov A.F., Ermakova G.V., Solovieva E.A., Lukyanov K.A., Bogdanova E.A., Zaraisky A.G., Lukyanov S., Chudakov D.M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4 (9), 741–6 [+]

    Разработан новый флуоресцентный белок Katushka, обладающий флуоресценцией в дальне-красной области спектра, которая является предпочтительной для анализа сигнала внутри тканей животных. Katushka в десять раз ярче, чем созданные ранее дальне-красные флуоресцентные белки и характеризуется высокой скоростью созревания, высокой рН-стабильностью и фотостабильностью. Это делает новый белок идеальным инструментом для прижизненного мечения клеток внутри целых организмов. Создан мономерный вариант белка Katushka, названный mKate, для исследования внутриклеточной локализации белков.

  14. Merzlyak E.M., Goedhart J., Shcherbo D., Bulina M.E., Shcheglov A.S., Fradkov A.F., Gaintzeva A., Lukyanov K.A., Lukyanov S., Gadella T.W., Chudakov D.M. (2007). Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4 (7), 555–7 [+]

    Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.

  15. Bulina M.E., Chudakov D.M., Britanova O.V., Yanushevich Y.G., Staroverov D.B., Chepurnykh T.V., Merzlyak E.M., Shkrob M.A., Lukyanov S., Lukyanov K.A. (2006). A genetically encoded photosensitizer. Nat. Biotechnol. 24 (1), 95–9 [+]

    Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to beta-galactosidase and phospholipase Cdelta1 pleckstrin homology domain.

  16. Chudakov D.M., Lukyanov S., Lukyanov K.A. (2005). Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23 (12), 605–13 [+]

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.

  17. Lukyanov K.A., Chudakov D.M., Lukyanov S., Verkhusha V.V. (2005). Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6 (11), 885–91 [+]

    The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.

  18. Chudakov D.M., Verkhusha V.V., Staroverov D.B., Souslova E.A., Lukyanov S., Lukyanov K.A. (2004). Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22 (11), 1435–9 [+]

    In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.

  19. Chudakov D.M., Feofanov A.V., Mudrik N.N., Lukyanov S., Lukyanov K.A. (2003). Chromophore environment provides clue to "kindling fluorescent protein" riddle. J. Biol. Chem. 278 (9), 7215–9 [+]

    asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

  20. Chudakov D.M., Belousov V.V., Zaraisky A.G., Novoselov V.V., Staroverov D.B., Zorov D.B., Lukyanov S., Lukyanov K.A. (2003). Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21 (2), 191–4 [+]

    Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.