About

Michael Caplow, PhD, is a professor in the Department of Biochemistry and Biophysics at the University of North Carolina at Chapel Hill. He earned his doctoral degree from Brandeis University. His research interests focus on the biochemistry of the cytoskeleton, specifically analyzing factors that stabilize the dimer interface in proteins involved in polymerization reactions. His work includes studying the tubulin α-β heterodimer, which forms microtubules essential for cell transport and division in all eukaryotes, and Cu,Zn superoxide dismutase 1 (SOD1), which forms protofibrils in familial amyotrophic lateral sclerosis (FALS). His research aims to understand the stability of these protein interfaces and their roles in cellular processes and disease mechanisms. Current projects involve examining how protein cofactors influence tubulin dimer assembly and stability, as well as investigating the kinetics of SOD1 aggregation and its implications in neurodegenerative diseases.

Research topics

• Biology
• Molecular biology
• Biochemistry
• Cell biology

Selected publications

• Kinetics and mechanism of microtubule length changes by dynamic instability.

  UNC Libraries · 2021-07-03

  articleOpen access

  Microtubules at steady state were found to undergo dramatic changes in length, with only very little change in number concentration and mean length. This result is accounted for by a mechanism in which microtubules are capped at ends by tubulin-GTP subunits; loss of the tubulin-GTP cap at one end results in disassembly of all the tubulin-GDP subunits, so that the medial edge of the distal tubulin-GTP cap is exposed; the exposed tubulin-GTP cap is sufficiently stable, so that microtubule regrowth from the cap rather than loss of the cap occurs. This mechanism predicts that a bell-shaped length distribution of sheared microtubules will be transiently bimodal, with peaks of short and moderate length microtubules, in rearranging to an exponential length distribution. We have observed the predicted transient bimodal length distribution experimentally and in a Monte Carlo simulation. Dynamic instability has recently been accounted for by assuming that microtubule ends are capped with only a single tubulin-GTP subunit at each end of the five helices that serve as elongation sites. Such a minimal tubulin-GTP cap is apparently ruled out by our observations, which require that the remnant tubulin-GTP cap generated from disassembly be able to serve as nucleating site; we do not expect that a stable nucleating site can be generated from five tubulin-GTP subunits, oriented as the five helices that serve as elongation sites.

  PublisherDOI

• Mechanism of the microtubule GTPase reaction

  UNC Libraries · 2021-07-03

  articleOpen accessSenior author

  The rate of GTP hydrolysis by microtubules has been measured at tubulin subunit concentrations where microtubules undergo net disassembly. This was made possible by using microtubules stabilized against disassembly by reaction with ethylene glycol bis-(succinimidylsuccinate) (EGS) as sites for the addition of tubulin-GTP subunits. The tubulin subunit concentration was varied from 25 to 90% of the steady state concentration, and there was no net elongation of stabilized microtubule seeds. The GTPase rate with EGS microtubules was linearly proportional to the tubulin-GTP subunit concentration when this concentration was varied by dilution and by using GDP to compete with GTP for the tubulin E-site. The linear dependence of the rate is consistent with a GTP mechanism in which hydrolysis is coupled to the tubulin-GTP subunit addition to microtubule ends. It is inconsistent with reaction schemes in which: microtubules are capped by a single tubulin-GTP subunit, which hydrolyzes GTP when a tubulin-GTP subunit adds to the end; hydrolysis occurs primarily in subunits at the interface of a tubulin-GTP cap and the tubulin-GDP microtubule core; hydrolysis is not coupled to subunit addition and occurs randomly in subunits in a tubulin-GTP cap. It was also found that GDP inhibition of the microtubule GTPase rate results from GDP competition for GTP at the tubulin subunit E-site. There is no additional effect of GDP on the GTPase rate resulting from exchange into tubulin subunits at microtubule ends.

  PublisherDOI

• Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules.

  UNC Libraries · 2020-11-01

  articleOpen access1st authorCorresponding

  Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly(catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCpP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 to 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of the rate constant for dissociation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microtubules, now found to be 0.118 s-1, has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as previously reported.

  PublisherDOI

• Glutathionylation at Cys-111 Induces Dissociation of Wild Type and FALS Mutant SOD1 Dimers

  Figshare · 2020-10-31

  articleOpen accessSenior author

  Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ~10−20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an ~30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.

  OA PDFDOI

• Non-native Soluble Oligomers of Cu/Zn Superoxide Dismutase (SOD1) Contain a Conformational Epitope Linked to Cytotoxicity in Amyotrophic Lateral Sclerosis (ALS)

  UNC Libraries · 2020-10-31

  articleOpen access

  Soluble misfolded Cu/Zn superoxide dismutase (SOD1) is implicated in motor neuron death in amyotrophic lateral sclerosis (ALS); however, the relative toxicities of the various non-native species formed by SOD1 as it misfolds and aggregates are unknown. Here, we demonstrate that early stages of SOD1 aggregation involve the formation of soluble oligomers that contain an epitope specific to disease-relevant misfolded SOD1; this epitope, recognized by the C4F6 antibody, has been proposed as a marker of toxic species. Formation of potentially toxic oligomers is likely to be exacerbated by an oxidizing cellular environment, as evidenced by increased oligomerization propensity and C4F6 reactivity when oxidative modification by glutathione is present at Cys-111. These findings suggest that soluble non-native SOD1 oligomers, rather than native-like dimers or monomers, share structural similarity to pathogenic misfolded species found in ALS patients and therefore represent potential cytotoxic agents and therapeutic targets in ALS.

  PublisherDOI

• Modifications of Superoxide Dismutase (SOD1) in Human Erythrocytes: A POSSIBLE ROLE IN AMYOTROPHIC LATERAL SCLEROSIS

  UNC Libraries · 2020-11-09 · 7 citations

  articleOpen access1st authorCorresponding

  Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation.

  PublisherDOI

• ATP Mediates Yeast Cell-Cell Communication

  bioRxiv (Cold Spring Harbor Laboratory) · 2020

  1st authorCorresponding
  • Biology
  • Cell biology
  • Molecular biology

  Abstract Yeast secrete ATP in response to glucose, a property with previously unknown functional consequence. In this report, we show that extracellular ATP is a signal for growth of surrounding cells. The ATP signaling behavior was serendipitously uncovered by finding reduced toxicity of an inducible, dominant-lethal form of alpha tubulin (tub1-828); lethality was dramatically reduced in cultures grown at high, compared to low cell density. Reduced cell death at high cell density occurred because the mutant tubulin’s deleterious effect of mitosis was reduced such that the rate of chromosome loss/cell division was lower (18-fold) in cultures inoculated with a high density (75,000/ml) compared to a low density (1,000/ml) of cells. The sparing effect of growth at high cell density could be replicated by co-culturing a low number of cells (3440/5 ml) that expressed tub1-828, with a high number of cells (2.3 E6/5 ml) that did not express the mutant protein. In this condition toxicity was reduced at high cell density apparently because it produced a sufficient concentration of a secreted growth substance, such that the mutant protein was rapidly diluted by synthesis of wild-type alpha tubulin (TUB1). Enhanced growth at high cell density was confirmed by fluorescence-activated cell sorting (FACS) analysis after DNA staining, which showed that the rate of the G1-G2 transition was faster for cells at high density. ATP replaced the need for high cell density for resistance to tub1-828, and stimulated the transition from G1 to G2 in cells at low density. This newly discovered quorum sensing response in yeast, mediated by ATP, indicates that yeast decision-making is not entirely autonomous.

  PublisherOA PDFDOI

• Ubiquitin editing enzyme UCH L1 and microtubule dynamics: Implication in mitosis

  UNC Libraries · 2020-11-01

  articleOpen access

  Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, western blot analysis of tubulin fractions after polymerization revealed the presence of a specific ∼50 kDa band of UCH L1 (not the normal ∼25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. the inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the ∼50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.

  PublisherDOI

• Dissociation of the Tubulin Dimer Is Extremely Slow, Thermodynamically Very Unfavorable, and Reversible in the Absence of an Energy Source

  UNC Libraries · 2020-11-01

  articleOpen access1st authorCorresponding

  The finding that exchange of tubulin subunits between tubulin dimers (α-β + α′β′ ↔ α′β + αβ′) does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the Kd for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was ∼9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10−11 M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 μM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10−11 M Kd was obtained from gel exclusion chromatography studies of 0.02–2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.

  PublisherDOI

• Jan Hermans (1933‐2018): Red‐blooded biophysicists study hemoglobin

  Proteins Structure Function and Bioinformatics · 2019-02-19

  article
  PublisherDOI

Recent grants

• NIH Grant R01GM046773

  NIH · $691k · 1998

• NIH Grant R01DE003246

  NIH · $923k · 1991

• NIH Grant R01GM059231

  NIH · $846k · 2005

Frequent coauthors

• Barry R. Zeebèrg

  16 shared

• John Shanks

  Emory University

  15 shared

• Nikolay V. Dokholyan

  Hershey (United States)

  14 shared

• Rachel L. Redler

  Johns Hopkins University

  8 shared

• Lanette Fee

  Duke Medical Center

  7 shared

• William P. Jencks

  5 shared

• Bruna P. Brylawski

  5 shared

• Kyle C. Wilcox

  AbbVie (United States)

  5 shared

Labs

• UNC Department of Biochemistry and BiophysicsPI

Education

• Ph.D.

  Brandeis University