Phosphorylation impacts GLE1 nuclear localization and association with DDX1.

Gle1 regulates gene expression at multiple steps from transcription to mRNA export to translation under stressed and non-stressed conditions. To better understand Gle1 function in stressed human cells, specific antibodies were generated that recognized the phosphorylation of threonine residue 102 (T102) in Gle1. A series of in vitro kinase assays indicated that T102 phosphorylation serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster. Indirect immunofluorescence microscopy with the anti-Gle1-pT102 antibodies revealed that basally phosphorylated Gle1 was pre-dominantly nuclear with punctate distribution; however, under sodium arsenite-induced stress, more cytoplasmic localization was detected. Immunoprecipitation with the anti-Gle1-pT102 antibody resulted in co-isolation of Gle1-pT102 with the DEAD-box protein DDX1 in a phosphatase sensitive manner. This suggested Gle1 phosphorylation might be linked to its role in regulating DDX1 during transcription termination. Notably, whereas the total Gle1-DDX1 association was decreased when Gle1 nucleocytoplasmic shuttling was disrupted, co-isolation of Gle1-pT102 and DDX1 increased under the same conditions. Taken together, these studies demonstrated that Gle1 phosphorylation impacts its cellular distribution and potentially drives nuclear Gle1 functions in transcription termination. We propose a model wherein phosphorylation of Gle1 either reduces its nucleocytoplasmic shuttling capacity or increases its binding affinity with nuclear interaction partners.

Assessing State Variation in Plastic Surgeons' Risk of Hepatitis C Exposure: Revisit in Methodology.

BACKGROUND: As the national opioid epidemic escalates, rates of the Hepatitis C (HCV) infection have similarly risen. Surgeons exposed intraoperatively secondary to sharp instrument or needle-sticks are affected both socioeconomically and physically. Current treatment strategies involve antiretroviral agents that have not been universally available. This study evaluates the current risk of surgeon exposure to HCV. METHODS: CDC data regarding state-by-state HCV diagnosis reporting were combined with the plastic surgery workforce data from the ASPS. Proxy variables for exposure risk to HCV were generated for each state and compared. RESULTS: West Virginia plastic surgeons were found to have a significantly elevated risk of exposure (60.0 versus 18.7, P < 0.0001). Their exposure risk is a notable outlier compared with the rest of the country (Risk >3 × IQR + 75th percentile). Similarly, states within the Ohio Valley were found to be at increased risk (34.8 versus 16.0, P = 0.05). States most heavily burdened by the opioid crisis were found to be at an increased risk for HCV exposure (40.8 versus 13.6, P = 0.0003). CONCLUSIONS: Plastic surgeons employed in states within the Ohio Valley were found to be at an increased risk of exposure to HCV. Plastic surgeons operating in states severely impacted by the opioid crisis were found to be at an increased risk of exposure. These findings underscore the importance of reducing the risk in the operating room and the need for better data collection to better understand this association and mitigate the risk to the operating surgeon.

Functions of Gle1 are governed by two distinct modes of self-association.

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

MAPK- and glycogen synthase kinase 3-mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.

Rapid expression of critical stress response factors is a key survival strategy for diseased or stressed cells. During cell stress, translation is inhibited, and a pre-existing pool of cytoplasmic mRNA-protein complexes reversibly assembles into cytoplasmic stress granules (SGs). Gle1 is a conserved modulator of RNA-dependent DEAD-box proteins required for mRNA export, translation, and stress responses. Proper Gle1 function is critical as reflected by some human diseases such as developmental and neurodegenerative disorders and some cancers linked to gle1 mutations. However, the mechanism by which Gle1 controls SG formation is incompletely understood. Here, we show that human Gle1 is regulated by phosphorylation during heat shock stress. In HeLa cells, stress-induced Gle1 hyperphosphorylation was dynamic, primarily in the cytoplasmic pool, and followed changes in translation factors. MS analysis identified 14 phosphorylation sites in the Gle1A isoform, six of which clustered in an intrinsically disordered, low-complexity N-terminal region flanking the coil-coiled self-association domain. Of note, two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), phosphorylated the Gle1A N-terminal domain, priming it for phosphorylation by glycogen synthase kinase 3 (GSK3). A phosphomimetic gle1A6D variant (in which six putative Ser/Thr phosphorylation sites were substituted with Asp) perturbed self-association and inhibited DEAD-box helicase 3 (X-linked) (DDX3) ATPase activity. Expression of alanine-substituted, phosphodeficient GFP-gle1A6A promoted SG assembly, whereas GFP-gle1A6D enhanced SG disassembly. We propose that MAPKs and GSK3 phosphorylate Gle1A and thereby coordinate SG dynamics by altering DDX3 function.

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA-dependent DEAD-box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP6 )-bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure-function analysis in S. cerevisiae and human (h) cells, and find that the high-affinity Nup42-Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy-terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5/hDDX19B interaction site. A nup42-CTD/gle1-CTD/Dbp5 trimeric complex forms in the presence of IP6 . Deletion of NUP42 abrogates Gle1-Dbp5 interaction, and disruption of the Nup42 or IP6 binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42-hGle1B interaction.

Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3).

DNA damage and other forms of replication stress can cause replication forks to stall. Replication stress response proteins stabilize and resolve stalled forks by mechanisms that include fork remodeling to facilitate repair or bypass of damaged templates. Several enzymes including SMARCAL1, HLTF, and ZRANB3 catalyze these reactions. SMARCAL1 and HLTF utilize structurally distinct accessory domains attached to an ATPase motor domain to facilitate DNA binding and catalysis of fork remodeling reactions. Here we describe a substrate recognition domain within ZRANB3 that is needed for it to recognize forked DNA structures, hydrolyze ATP, catalyze fork remodeling, and act as a structure-specific endonuclease. Thus, substrate recognition domains are a common feature of fork remodeling, SNF2-family, DNA-dependent ATPases, and our study provides further mechanistic understanding of how these enzymes maintain genome integrity during DNA replication.

A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.

SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1(CD)) is composed of an SNF2-type double-stranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1(CD). Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain's role in replication fork stability and genome integrity.

Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1-RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 µM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.

Substrate-selective repair and restart of replication forks by DNA translocases.

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.

SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1) maintains genome integrity during DNA replication. Here we investigated its mechanism of action. We found that SMARCAL1 travels with elongating replication forks, and its absence leads to MUS81-dependent double-strand break formation. Binding to specific nucleic acid substrates activates SMARCAL1 activity in a reaction that requires its HARP2 (Hep-A-related protein 2) domain. Homology modeling indicates that the HARP domain is similar in structure to the DNA-binding domain of the PUR proteins. Limited proteolysis, small-angle X-ray scattering, and functional assays indicate that the core enzymatic unit consists of the HARP2 and ATPase domains that fold into a stable structure. Surprisingly, SMARCAL1 is capable of binding three-way and four-way Holliday junctions and model replication forks that lack a designed ssDNA region. Furthermore, SMARCAL1 remodels these DNA substrates by promoting branch migration and fork regression. SMARCAL1 mutations that cause Schimke immunoosseous dysplasia or that inactivate the HARP2 domain abrogate these activities. These results suggest that SMARCAL1 continuously surveys replication forks for damage. If damage is present, it remodels the fork to promote repair and restart. Failures in the process lead to activation of an alternative repair mechanism that depends on MUS81-catalyzed cleavage of the damaged fork.

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

Functions of alternative replication protein A in initiation and elongation.

Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair, and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative replication protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to those of the canonical RPA complex but does not support DNA replication. We describe studies that aimed to identify what properties of aRPA prevent it from functioning in DNA replication. We show aRPA has weakened interaction with DNA polymerase alpha (pol alpha) and that aRPA is not able to efficiently stimulate DNA synthesis by pol alpha on aRPA-coated DNA. Additionally, we show that aRPA is unable to support de novo priming by pol alpha. Because pol alpha activity is essential for both initiation and Okazaki strand synthesis, we conclude that the inability of aRPA to support pol alpha loading causes aRPA to be defective in DNA replication. We also show that aRPA stimulates synthesis by DNA polymerase alpha in the presence of PCNA and RFC. This indicates that aRPA can support extension of DNA strands by DNA polymerase partial differential. This finding along with the previous observation that aRPA supports early steps of nucleotide excision repair and recombination indicates that aRPA can support DNA repair synthesis that requires polymerase delta, PCNA, and RFC and support a role for aRPA in DNA repair.

An alternative form of replication protein a expressed in normal human tissues supports DNA repair.

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.

FANCJ helicase uniquely senses oxidative base damage in either strand of duplex DNA and is stimulated by replication protein A to unwind the damaged DNA substrate in a strand-specific manner.

FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism.

Reagentless, electrochemical approach for the specific detection of double- and single-stranded DNA binding proteins.

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.

An alternative form of replication protein a prevents viral replication in vitro.

Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting "alternative" RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the canonical RPA complex; however, aRPA is unable to support DNA replication and inhibits canonical RPA function. Two regions of RPA4, the putative L34 loop and the C terminus, are responsible for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA function in vitro and is found in nonproliferative tissues, these studies indicate that RPA4 expression may prevent cellular proliferation via replication inhibition while playing a role in maintaining the viability of quiescent cells.

Cellular functions of human RPA1. Multiple roles of domains in replication, repair, and checkpoints.

In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.

Protein-protein binding is often associated with changes in protonation state.

pK(a) values of ionizable residues have been calculated using the PROPKA method and structures of 75 protein-protein complexes and their corresponding free forms. These pK(a) values were used to compute changes in protonation state of individual residues, net changes in protonation state of the complex relative to the uncomplexed proteins, and the correction to a binding energy calculated assuming standard protonation states at pH 7. For each complex, two different structures for the uncomplexed form of the proteins were used: the X-ray structures determined for the proteins in the absence of the other protein and the individual protein structures taken from the structure of the complex (referred to as unbound and bound structures, respectively). In 28 and 77% of the cases considered here, protein-protein binding is accompanied by a complete (>95%) or significant (>50%) change in protonation state of at least one residue using unbound structures. Furthermore, in 36 and 61% of the cases, protein-protein binding is accompanied by a complete or significant net change in protonation state of the complex relative to the separated monomers. Using bound structures, the corresponding values are 12, 51, 20, and 48%. Comparison to experimental data suggest that using unbound and bound structures lead to over- and underestimation of binding-induced protonation state changes, respectively. Thus, we conclude that protein-protein binding is often associated with changes in protonation state of amino acid residues and with changes in the net protonation state of the proteins. The pH-dependent correction to the binding energy contributes at least one order of magnitude to the binding constant in 45 and 23%, using unbound and bound structures, respectively.

The cellular plasticity of human adipocytes.

Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.