Integrative and Comparative Biology
Integrative and Comparative Biology, volume 52, number 5, pp. 665–680
doi:10.1093/icb/ics058
Society for Integrative and Comparative Biology
SYMPOSIUM
Proteomic Responses of Sea Urchin Embryos to Stressful
Ultraviolet Radiation
N. L. Adams,*,1 J. P. Campanale† and K. R. Foltz‡
*California Polytechnic State University, San Luis Obispo, CA, USA; †Scripps Institution of Oceanography, University of
California, San Diego, CA, USA; ‡University of California, Santa Barbara, CA, USA
From the symposium ‘‘Comparative Proteomics of Environmental and Pollution Stress’’ presented at the annual meeting
of the Society for Integrative and Comparative Biology, January 3–7, 2012 at Charleston, South Carolina.
1
E-mail: [email protected]
Synopsis Solar ultraviolet radiation (UVR, 290–400 nm) penetrates into seawater and can harm shallow-dwelling and
planktonic marine organisms. Studies dating back to the 1930s revealed that echinoids, especially sea urchin embryos, are
powerful models for deciphering the effects of UVR on embryonic development and how embryos defend themselves
against UV-induced damage. In addition to providing a large number of synchronously developing embryos amenable to
cellular, biochemical, molecular, and single-cell analyses, the purple sea urchin, Strongylocentrotus purpuratus, also offers
an annotated genome. Together, these aspects allow for the in-depth study of molecular and biochemical signatures of
UVR stress. Here, we review the effects of UVR on embryonic development, focusing on the early-cleavage stages, and
begin to integrate data regarding single-protein responses with comprehensive proteomic assessments. Proteomic studies
reveal changes in levels of post-translational modifications to proteins that respond to UVR, and identify proteins that
can then be interrogated as putative targets or components of stress-response pathways. These responsive proteins are
distributed among systems upon which targeted studies can now begin to be mapped. Post-transcriptional and translational controls may provide early embryos with a rapid, fine-tuned response to stress during early stages, especially during
pre-blastula stages that rely primarily on maternally derived defenses rather than on responses through zygotic gene
transcription.
Introduction
Solar ultraviolet radiation (UVR) has been known to
negatively effect organisms since the late 19th century
(Downes and Blunt 1877). Studies in the early 1900s
revealed that exposure of echinoderm eggs and embryos to UVR resulted in dose-dependent delays in
cell division (Giese 1938; Blum et al. 1954) and subsequently characterized the morphological abnormalities
UVR caused in echinoderm larvae, including death
(Giese 1964; Rustad 1964). Today, echinoderms
remain the focus of many reports on the developmental
and physiological effects of UVR (reviewed by Dahms
and Lee 2010; Lamare et al. 2011). The technology used
to examine the molecular, cellular, and biochemical
nature of UV-induced effects, which include linking
UV-induced damage with specific proteins that function in ameliorating UV-stress to specific cellular pathways, has been available only recently.
While there have been reports evaluating effects of
UVR on DNA, and the response of specific transcripts, only a few reports have identified UVresponsive proteins (Dahms and Lee 2010; Lamare
et al. 2011). Many insights have been gained from
the large body of knowledge relating to effects of
UVR on proteins in mammalian cells, but a comprehensive examination of proteins affected by UVR in
sea urchin embryos has been lacking. Recently, a
comparative proteomic analysis of the embryos of
Strongylocentrotus purpuratus (California purple sea
urchin) revealed a number of proteins that respond
to UVR during the first mitotic cleavage
(Campanale et al. 2011). Here, we collectively evaluate these data to decipher clues about the molecular
mechanisms of UVR-induced damage, defensive
mechanisms, and the cellular basis controlling responses to UVR-stress.
Advanced Access publication May 10, 2012
ß The Author 2012. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
666
Characterization of ultraviolet radiation
in the marine environment: From
molecules, to organisms, to ecosystems
Solar UVR reaching the Earth’s surface (UVR:
290–400 nm) significantly penetrates into seawater and
can harm marine organisms (Bancroft et al. 2007; Häder
et al. 2011). Ultraviolet A (UV-A) wavelengths
(320–400 nm) are longer and penetrate deeper than do
those of ultraviolet B (UV-B; 290–320 nm; Smith and
Baker 1979); however, UV-B is more biologically damaging than UV-A (Tevini 1993). Penetration of UV-B
wavelengths has increased due to ozone depletion
(Madronich 1998; WMO 2007) and despite global reductions in the release of ozone-depleting gases and stabilization of ozone levels, UV-B irradiance is predicted to
increase by a few percent per decade due to global climatic change (McKenzie et al. 2003, 2007; WMO 2007).
Similarly, penetration of UV in coastal oceans is expected to increase due to global warming and acidification of seawater. Increased UV-irradiation can
photobleach UV-absorbing chromophoric dissolved organic matter (CDOM) and increase penetration of UV
into the water column, leading to synergistic stresses in
marine communities (Whitehead et al. 2000; Przeslawski
2005; UNEP 2005; Häder et al. 2011; Zepp et al. 2011).
UV-B causes direct damage to proteins, DNA, and
membrane lipids. In addition to photochemical degradation of proteins, UV-B directly damages DNA
through the production of cyclobutane pyrimidine
dimers (CPDs) and other photoproducts (Vincent and
Neale 2000; Sinha and Häder 2002). Reactive oxygen
species (ROS) generated by UV-A, rapidly oxidize
nucleic acids, proteins, and lipids (Tyrell 1991; Cadet
et al. 2003; Lesser 2006). This UV-induced molecular
damage is manifested as physiological stress, fundamentally affecting survivorship of embryonic, and larval
stages that live at the ocean’s surface (Gleason and
Wellington 1996; Przeslawski et al. 2005; Lamare et al.
2011; Häder et al. 2011). Eventually, these stressors delay
development and increase mortality both directly and
indirectly (Pechenik 1987; Worrest 1982; Karentz 1994;
Whitehead et al. 2000; Häder et al. 2011), and ultimately
alter trophic interactions and food webs (Bothwell et al.
1994; Caldwell et al. 1998; Mostajir et al. 2000; Häder
and Sinha 2005).
Sea urchins as a model for studying
effects of UVR
Echinoid embryos are well suited for quantitative
studies of UV-induced damage. Their gametes are
N. L. Adams et al.
easily obtained, fertilized, and cultured and they
have been used extensively as a model for elucidating
the effects of environmental factors such as temperature, oxidative stress, chemical pollutants, and UVR
since the early 1900s (Dahms and Lee 2010; Lamare
et al. 2011). Large eggs (80–200 mm diameter) allow
single-cell analyses, while abundant quantities of synchronously developing embryos facilitate molecular
and biochemical analyses. Like many organisms,
early sea urchin embryos rely on a robust pool of
mRNA and proteins provided maternally and that
control processes necessary for initiating and deploying the developmental program (Davidson et al.
1998; Davidson 2006a). Thus, the early sea urchin
embryo develops and responds to stressors primarily
through post-transcriptional and post-translational
mechanisms (reviewed by Epel 2003).
Additionally, the sequencing of the S. purpuratus
genome (Sea urchin Genome Consortium 2006;
Sodergren et al. 2006) and description of the developmental transcriptome (Samanta et al. 2006) led to
identification of the genes implicated in a variety of
biological processes (Davidson 2006b). Significantly,
the arsenal of gene products for chemical defense,
including oxidative stress proteins, are now annotated (Goldstone et al. 2006). These surveys of the
genome provide a platform for predicting and analyzing the proteins that are involved in responses to
stresses such as UVR.
UVR and sea urchin embryos
Developmental effects of UVR on sea urchin embryos
A large body of historical work documenting the effects
of UVR on sea urchin embryos and larvae serves as an
instructive base for molecular studies (reviewed by
Häder et al. 2011; Lamare et al. 2011). The effects of
UVR are both dose-dependent and wavelengthdependent. Sub-lethal doses lead to delays in development while higher doses or lower wavelengths arrest
development or cause-death (Fig. 1; Rustad 1971;
Adams and Shick 2001; Lamare et al. 2011). Experiments using controlled doses of environmentallyrelevant UVR have allowed for quantitative
assessments of developmental delays and qualitative
morphological aberrations (reviewed by Dahms and
Lee 2010; Lamare et al. 2011). Furthermore, in situ
studies demonstrate that both solar UV-A and UV-B
induce developmental abnormalities in both polar and
temperate sea urchin species (Lamare et al. 2011). To
assess the effects of UV-A and UV-B independently,
Proteomic responses of sea urchin embryos
667
Fig. 1 Sea urchin embryos treated with UVR (290–400 nm) experience delays and defects in development. Embryos protected from
UVR undergo normal development, including cell division during the early-cleavage stages, eventually transitioning from the swimming
nonfeeding blastula through gastrulation to a feeding pluteus. Treatment immediately after fertilization with sub-lethal doses of UVR
(290–400 nm from QPanel UV340 lamps) caused delays in cell division during the early-cleavage stages, while chronic irradiation
(6 h/day from fertilization) results in abnormal development. Cells ‘‘pack’’ the blastocoel of blastulae, embryos exogastrulate, and
pluteus larvae are deformed. Modified with permission from Adams and Shick (2001).
Fig. 2A shows the spectral irradiance of solar UVR that
penetrates into surface waters in Central California
through filters designed to expose or protect embryos
from UVR. Using this experimental design, we have
observed that embryos of S. purpuratus display the
characteristic dose-dependent delay in cleavage when
exposed to UVR in situ (Fig. 2B).
Nonprotein defensive strategies
Motile (later stage) sea urchin embryos have defensive strategies that guard against UVR damage,
including avoiding light if held in static water
(Miller and Emlet 1997). Nevertheless, these larvae
may not be able to control their position in a mixed
water column (Denny and Shibata 1989), possibly
leaving them more vulnerable to sustained exposure
to UVR. Sea urchin embryos also accumulate natural
sunscreens, mycosporine-like amino acids (MAAs)
that are provided maternally to eggs through the
adult’s diet (Carroll and Shick 1996) and that protect
embryos against UVR-induced cleavage delays in
cleavage and against developmental abnormalities
(Adams and Shick 1996, 2001; Lamare et al. 2011).
However, adult S. purpuratus have relatively low and
variable concentrations of MAAs in their gonads,
epidermis, and gametes (Gravem 2009). Hoffman
and Lamare (2004) observed similar results and emphasized that other modes of protection, such as carotenoids, may be more important. These and other
nonprotein defenses were reviewed recently by
Lamare et al. (2011), whereas the remainder of our
review will focus on UV-induced changes in proteins.
Fig. 2 In situ measurements and effects of solar radiation at the
Center for Coastal Marine Sciences Pier facility in San Luis Bay,
CA, USA. (A) Average surface irradiance through UV-opaque
(UVO) and UV-transparent (UVT) acrylic during three different
sunny days in the summer of 2008. Measurements were taken
using an OL756 spectroradiometer (Photonics) and reveal the
differences in UVR between two in situ treatments: protected
from solar UVR (UVO) or treated with full spectrum UVR
(UVT). (B) Cleavage rate of newly fertilized embryos exposed to
(gray line) or protected from (black line) natural UVR at 1 m
depth for 30 min in situ using the design described in A.
668
Overview of cellular and molecular
responses of early sea urchin embryos
to UVR
The cellular stress response
Sea urchin embryos actively deploy a variety of
defensive mechanisms that continue embryogenesis
in stressful environments, including selective packaging of defensive proteins that combat toxic environmental chemicals, including heavy metals, and during
periods of redox flux (Hamdoun and Epel 2007).
Exposure to wavelengths between 320 and 500 nm
also increases photolyase activity, allowing repair of
CPDs (cf. Akimoto and Shiroya 1987a). Photolyase
transcription can be upregulated in blastula-stage embryos after exposure to UVR (Isely et al. 2009), but
studies of the production and regulation of photolyase
protein in sea urchins are lacking.
In addition, cellular stress responses (CSRs) sense
damage and often lead to delays in the cell cycle. All
organisms studied to date appear to activate the CSR
upon macromolecular damage during exposure to natural stressors, including UVR (Kültz 2005). The CSR
mobilizes multiple signaling pathways that ameliorate
molecular damage using primarily two mechanisms.
First, cells activate defensive proteins, including heatshock proteins (HSPs) that aid in folding and/or degrading proteins (Gething and Sambrook 1992; Feder and
Hofmann 1999). Second, cells delay their developmental
programs using kinases, such as check point kinases and
cyclin-dependant kinases, that control passage through
phases of the cell cycle and aid in providing time to
respond to macromolecular damage before undergoing
cell division (Kültz 2005).
Post-translational modifications and the responses to
UV-stress
Responses to stress often involve coordinated cellular
signaling pathways, including those that control the
cell cycle, metabolism, transcription, development and
differentiation, and apoptosis (Kültz 2005). Frequently,
the proteins that control these processes are regulated
by means of post-translational modifications (PTMs).
Phospohorylation is a well-documented PTM (cf
Cowan and Storey 2003), and only recently do we
have the technology to both map and understand the
importance of PTMs on a proteomic scale (Mann and
Jensen 2003); this has not been broadly applied yet to the
UVR stress responses such as delays in development.
The molecular pathways controlling cell division
and the regulation of Ca2þ in cells are highly conserved
N. L. Adams et al.
among organisms, including sea urchins (FernandezGuerra et al. 2006; Roux et al. 2006; Whitaker 2006).
Dynamic protein phosphorylation is involved in the
regulation of the signaling cascades for these processes
(Kalume et al. 2003; Roux et al. 2006). Many directed
studies, as well as the analysis of the S. purpuratus
genome, have further characterized the rich array of
protein kinases (Bradham et al. 2006) and phosphatases
(Byrum et al. 2006) available during development. The
egg and early embryo phosphoproteome is quite
dynamic (Roux et al. 2008). Analysis of the sea urchin
embryonic kinome revealed that 76% of kinases are
expressed during embryogenesis (Bradham et al.
2006). Thus, it is plausible that in sea urchin eggs and
early embryos, kinases (and attendant phosphatases)
regulate pathways that are poised to respond to stressors such as UVR.
While phosphorylation is a well-studied PTM, the
acetylation of proteins is also emerging as an important
PTM, regulating many signaling mechanisms and affecting protein function (Polevoda and Sherman 2002).
One example is the acetylation of amino-terminal peptide residues of proteins by sirtuins, which are
NAD-dependent deacetylases. Sirtuins can be induced
during hypoxia (Majmundar et al. 2010), are implicated in repair mechanisms, and appear to play a role
in activation of the heat-shock response (Westerheide
et al. 2009; Tomanek and Zuzow 2010). Further, microinjection of active sirtuins slows maturation and cell
division in sea star eggs and embryos, indicating a possible regulatory role for acetylation in controlling
timing of the cell cycle (Borra et al. 2002). At least
two forms of sirtuins have been identified in the sea
urchin genome, but no studies have yet examined protein acetylation in response to stress, such as UVR.
Ubiquitylation is another important regulatory PTM
(Vucic et al. 2011). The ubiquitin/proteasome pathway
is the major proteolytic system in eukaryotic cells for
selective degradation of short-lived regulatory proteins
(Ciechanover 1998; Ciechanover and Schwartz 1998).
Ubiquitin has been identified in sea urchin embryos
(Gong et al. 1991), but to our knowledge, there are no
published reports documenting effects of UVR on ubiquitylation in sea urchins, although protein turnover
and proteasome responses are evident (Campanale
et al. 2011; see below). Along with phosphorylation
and acetylation—as well as other PTMs not discussed
here—ubiquitylation could serve as a rapid response to
UVR damage.
669
Proteomic responses of sea urchin embryos
The proteomic perspective
Proteomic approaches have the ability to objectively
identify extensive responses by proteins to stress
(Aiken 2011; Tomanek 2011). Some of the earliest
studies of whole-embryo proteomes were conducted
in sea urchins (Brandhorst 1976; Grainger et al. 1986)
and there have also been proteomic studies probing the
responses to and recovery from UVR (cf. Akimoto and
Shiroya 1987a, 1987b). However, early studies were
limited by the inability to identify proteins. With the
annotation of the S. purpuratus genome and the power
to identify proteins using mass spectrometry, this approach has gained new traction.
In sea urchin embryos, at least several thousand different proteins are represented in the unfertilized egg
and a large percentage of these proteins turn over or
exhibit dynamic phosphorylation in just the first few
minutes post-fertilization (Roux et al. 2006, 2008;
Campanale et al. 2011). Proteomic studies are also
being employed to assess specific cell types and subcellular components (cf Mann et al. 2010), as well as the
immune response of sea urchins to pathogenic bacterial
infection (Dheilly et al. 2011). Furthermore, proteomic
approaches evaluating redox responses to environmental pollutants have just been commissioned in a variety
of model systems (Braconi et al. 2011). As technologies
for exploring proteomes advance (cf Altelaar and Heck
2012), it is increasingly clear that this approach
provides a powerful assessment of responses to stress.
Recently, a comparative proteomic assessment using
2DE followed by mass spectrometry identified UVRresponsive proteins in cleavage-stage embryos of
S. purpuratus. A number of these proteins are members
of the CSR, the kinome, and the proteasome, but many
proteins had not been identified as responding to UVR,
including the proteins controlling cellular metabolism
(Campanale et al. 2011). Many of the differences
between UV-protected and treated embryos observed
in that study may be due to changes in protein levels or
to PTMs (Fig. 3A), and represent particularly interesting proteins that we propose are part of the altered, or
induced, cellular signaling pathways (Fig. 3B) responding to sub-lethal UVR stress.
Specific protein responses of early
embryos to UVR
Changes in protein abundance or PTM serve as a signature of responsiveness to UVR treatment.
Comparative proteomic surveys serve as screens for
proteins that can then be investigated in more detail
for roles in defense, recovery, or even as direct targets.
UVR-responsive proteins identified in the proteomics
screen (Campanale et al. 2011) were subjected to analysis through BioSystems (Geer et al. 2010) to begin
mapping the UV-response pathways; then, targeted
studies identifying individual proteins were added.
Table 1 lists the biological pathways that are responsive
to UVR and a subset of these are described in more
detail below. Interestingly, assessing the collection of
responsive proteins reveals critical proteins that intersect various pathways or systems, such as the cell cycle,
translational control, and apoptosis, as well as conserved CSR pathways.
Cell division and apoptosis
In all eukaryotes, progression of the cell cycle is mediated by a series of phosphorylation and dephosphorylation events, as well as by formation and degradation
of protein complexes. Entry into M-phase is regulated
by mitosis-promoting factor (MPF), a complex of
cyclin and a cyclin-dependent kinase, Cdc2. Activation
of MPF by dephosphorylation of a regulatory tyrosine
in Cdc2 allows entry into M-phase. Exit from M-phase
requires the degradation of cyclin, mediated through
ubiquitylation (Murray and Kirschner 1989). Vital
checkpoints serve to ensure that replication of DNA
is faithfully completed and that chromosomes are
aligned and segregating properly. These checkpoints,
often involving sensor and effector kinases, modulate
cross talk between the mitotic spindle, pathways of
DNA damage and repair, and apoptosis. Ultimately,
they coordinate whether a cell will continue dividing
or undergo cell death (Tanaka 2010). All of these pathways are UVR-responsive (Table 1).
Because UVR is known to cause damage to DNA and
to arrest progression of the cell cycle in prophase
(Rustad 1964; Adams and Shick 1996; Lesser 2010;
Lamare et al. 2011; Fig. 2), it is not surprising that
many cell-cycle proteins are responsive to UVR. For
example, 14-3-3 proteins mediate progression of the
cell cycle through interaction with Cdc25, the phosphatase that activates MPF by dephosphorylating Cdc2
(Borgne and Meijer 1996). The 14-3-3 proteins regulate
pathways by binding to, and sequestering, clientsignaling proteins such as kinases and phosphatases
(Fu et al. 2000; Ferl et al. 2002). In mammalian cells,
UVR treatment results in checkpoint kinase (p38)dependent phosphorylation of Cdc25 as a response to
DNA damage. This initiates the binding of Cdc25 to
14-3-3, sequestering it and preventing MPF activation
and entry into M-phase (Bulavin et al. 2001).
670
N. L. Adams et al.
Fig. 3 (A) Comparative proteome map of protected and UVR-treated Strongylocentrotus purpuratus embryos at 30 min post-fertilization.
Fertilized eggs were protected from or exposed to UVR (2.125 J/cm2) for 30 min using a QPanel UV340 lamp (290–400 nm) and then
lysed and analyzed by 2D SDS–PAGE. A total of 100 g of TX-soluble protein was used for each gel and isoelectric focusing was for pH
range 3–7. The overlay (composite) of fusion-averaged 2D SDS–PAGE gel images for six batches of embryos is shown. Blue spots are
proteins from UV-protected embryos and orange spots represent UV-treated proteins. Black spots indicate overlap between the two.
Highlighted and numbered protein spots correspond to proteins that migrate differently due to UVR treatment and were identified by
MALDI-TOF MS þ MS/MS. With permission from Campanale et al. (2011). (B) Pie chart representing the relative proportion of
proteins in embryos of sea urchins affected by exposure to sub-lethal UVR in specific cellular pathways (functional categories). Proteins
that were affected by UVR in sea urchin embryos at 30 and 90 min post-fertilization were identified by mass spectrometry. Positively
identified proteins were then grouped into twenty functional categories based on eukaryotic clusters of orthologous groups (KOG).
Consistent with this, UV-irradiation of blastulae and
later-stage sea urchin embryos causes an increase in,
and anomalous patterns of, 14-3-3 " transcription
(Russo et al. 2010). Campanale et al. (2011) identified
14-3-3 proteins as UV-responsive during the first cell
cycle in sea urchin embryos (Table 1). In particular,
each of these proteins shifted in molecular weight
after UV-treatments (Fig. 4), suggestive of a PTM.
671
Proteomic responses of sea urchin embryos
Table 1 BioSystems (pathways) and proteins in each BioSystem responsive to UVR in early-cleavage stage sea urchin embryos
BioSystems
Example proteins
References
Apoptotic signaling
p53, p38MAPK, cyclophilin D
Campanale et al. (2011), Bonaventura et al. (2005)a,
Lesser et al. (2003)
Cell cycle control
Cdc2, p53, p21, 14-3-3, pRB, Vasa
Campanale et al. (2011), Bonaventura et al. (2005)a,
Lesser et al. (2003), Adams and Foltz (2002)
Biogenesis
UTP-glucose-1-phosphate uridylyltransferase,
YP30, PTK9L, SM30
Campanale et al. (2011), Bonaventura et al. (2005)a
Cytoskeleton
Actin, tubulin, tropomyosin, gelsolin
Campanale et al. (2011), Akimoto and Shiroya (1987b)
DNA damage response and
repair
Hhr23
Campanale et al. (2011)
Stress response
p38 MAPK, 14-3-3 protein, carbonic anhydrase
Campanale et al. (2011), Bonaventura et al. (2005)a,
Lesser et al. (2003)
ROS response
Superoxide dismutase, glutathione, catalase, thioredoxin, cyclophilin D, Glutathione peroxidase
Campanale et al. (2011), Lister et al. (2010)a, Lesser
et al. (2003)
Protein turnover, chaperones
Hsp70, chaperonin, proteasome complex
components
Campanale et al. (2011), Bonaventura et al. (2005,
2006)a
Amino acids
Glutamine synthetase
Campanale et al. (2011)
Carbohydrates
Adenosine kinase A, fructose-1,6-bisphosphase,
transaldolase
Campanale et al. (2011)
Cell division and apoptosis
Cellular structure
CSR and macromolecular damage
Metabolism and transport
Coenzymes
Adenosylhomocysteinase
Campanale et al. (2011)
Energy production
Isocitrate dehydrogenase, malate dehydrogenase
Campanale et al. (2011)
Lipids
Apolipoprotein B, hydroxymethylglutaryl-CoA
synthase
Campanale et al. (2011)
Nucleotides
Bisphosphate nucleotidase, IMP cyclohydrolase, intermediate chain 1
Campanale et al. (2011)
Transcription and translation
Transcription, RNA processing
Nucleolin, vasa, Ruvb-like protein
Campanale et al. (2011)
Translation
Translation elongation factors, eIF2, eIF3, L10e/P0
Campanale et al. (2011)
BioSystems are groups of proteins that interact as defined by NCBI BioSystems database (http://www.ncbi.nlm.nih.gov/biosystems/).
Indicates protein response occurred any time from cleavage to post-blastula stage in these studies (whereas other studies specifically evaluated
cleavage-stage embryos). References describing transcriptional responses in later stage embryos are not included on this list.
a
The 14-3-3 proteins are regulated by phosphorylation
and acetylation (van Heusden 2009), indicating this
could partly provide a rapid response to UV by the
early embryo.
If DNA damage checkpoints are acting upstream
of 14-3-3 in early-cleavage sea urchin embryos, then
delayed activation of Cdc25, and thus Cdc2, could
partly explain the delay in the cell cycle. UVR causes
delays in dephosphorylation of Cdc2 at Tyr 15
(maintaining it in its inactive form), which delays
the first cell cycle (N. L. Adams, manuscript in
preparation). Lesser et al. (2003) observed increases
in levels of Cdc2 proteins in fertilized embryos with
increasing UVR wavelengths that triggered apoptosis,
suggesting that translation or turnover of Cdc2 may
be responsive to UV as well.
Reports of stress-induced apoptosis in early sea
urchin embryos have differed. Lesser et al. (2003)
observed UV-induced apoptosis and Voronina and
Wessel (2001) observed chemical-induced apoptosis
in sea urchin eggs, and early embryos. In contrast,
Vega and Epel (2004) reported that stress-induced
672
N. L. Adams et al.
acetylation and phosphorylation, and these also modulate assembly and stability of microtubules (Janke and
Bulinski 2011). Actin dynamics may be similarly affected by PTMs (Dominguez and Holmes 2011).
Gelsolin (regulated by tyrosine phosphorylation), a
regulator of actin polymerization/severing (Sun et al.
1999), Arp3, an actin-related protein, and F-actin capping protein all exhibited UVR-responsiveness (Table
1). Interestingly, disruption of actin cytoskeleton by
lethal doses of UVR or chemical disruptors can initiate
apoptosis in mammalian cells (Kulms et al. 2002; Levee
et al. 1996). Although these studies used UV-lamps that
had peak excitations in the UV-B spectrum and used
doses much higher than is observed in the environment, they set the stage for future investigations linking
UVR treatment with the cytoskeletal dynamics that interface with regulation of the cell cycle and potentially
control induction of apoptosis.
CSR and macromolecular damage
Fig. 4 Response of 14-3-3 protein to UVR. (A) Magnification of
spots from composite proteomes (Fig. 3) that were identified as
14-3-3 protein isoform 1. (B) Comparisons of density of spots
shown in A indicate UVR caused a significant difference in spot
density and a possible shift in pI (suggestive of a PTM; ANOVA,
P50.05).
apoptosis is rare in early sea urchin embryos and
more prevalent in post-blastula stages, in which transcription is occurring. It is possible that the differences among these studies are due to different
exposures or to the detection techniques and species
used. Campanale et al.’s (2011) proteomic screen of
UV-stressed cleavage-stage embryos identified only
proteins indirectly involved in apoptosis and in
crossover with other pathways (see ‘‘Cellular structure,’’ ‘‘CSR and macromolecular damage,’’ and
‘‘Translation’’ sections below).
Cellular structure
Studies in human skin cells indicate that natural UVR
affects regulation of the cytoskeleton (Zamanski and
Chou 1987). Campanale et al. (2011) identified potential UV-responsive cytoskeletal proteins in sea urchins,
including cytoskeletal regulators as well as actins and
tubulins (Table 1). UVR affected Rho-GDP dissociation inhibitor, which plays a role in cytokinesis by
promoting the growth of microtubules, especially
during interphase (Robinson and Spudich 2000).
Tubulins are subject to a variety of PTMs, including
Chaperone function
Molecular chaperones, called HSPs, are necessary for
protein folding and translocation (Gething and
Sambrook 1992) as well as for control of the cell cycle
(Helmbrecht et al. 2000; Geraci et al. 2003). Cells
constitutively express HSPs, and for marine invertebrates, expression is often tightly coupled to environmental gradients (Feder and Hoffman 1999; Tomanek
2010). Expression and function of HSP proteins can
change during development and after macromolecular
damage incurred by environmental stress, whereas interactions among HSPs and macromolecules sensitive
to UVR result in upregulation of HSP expression
(Feder and Hoffman 1999; Niu et al. 2006).
HSP70 in the sea urchin Paracentrotus lividus localizes
on the mitotic asters with Cdc2/Cyclin B in dividing
embryos (Geraci et al. 2003) and is required for
proper assembly of the mitotic asters during cell division
(Sconzo et al. 1999). Agueli et al. (2001) proposed that
HSP70 performs an essential chaperone role for the folding of tubulin during assembly of the embryo’s mitotic
apparatus in sea urchins. Abiotic stressors induce HSP70
transcription in later-stage sea urchin embryos, but not
in early-cleavage stages (which are relatively transcriptionally silent; Giudice et al. 1999) and UVR causes up
regulation of HSP70 transcripts and protein in the blastula stage of P. lividus embryos (Bonaventura et al. 2005,
2006). HSPs were also captured in the UV-responsive
proteomic screen of early embryos (Campanale et al.
2011; Table 1). UVR caused the pI of HSP70 to
673
Proteomic responses of sea urchin embryos
become more acidic compared to protected embryos at
30 min post-fertilization, possibly due to PTM (Fig. 5A
and B). In contrast to small increases in the concentration of HSP70 protein in early-cleavage stages observed
by Bonaventura et al. (2006), our recent studies in early
embryos of S. purpuratus reveal that the concentration
of HSP is not significantly affected by sub-lethal doses of
UVR (Fig. 5C and D). This difference may be due to
differences in species or in UV-dose. In addition to measuring spot densities and migration (Fig. 5A and B),
direct immuno-detection on 2D gels reveals that UVR
treatment of early embryos causes changes in the HSP 70
pI, indicating a potential PTM of HSP 70 (Fig. 5D).
Collectively, the directed and proteomic studies support
a model in which HSPs are part of a UVR stress response
in early sea urchin embryos, primarily through PTMs.
Oxidative stress pathways
Shortly after fertilization, a respiratory burst transitioning a metabolically quiescent egg to a rapidly dividing
embryo results in the production of ROS (Shapiro
1991; Wong et al. 2004). Many studies have shown
that oxidative pathways are affected by UVR in sea
urchin embryos, indicating that superoxide dismutase
(SOD) is upregulated in response to UVR (Lesser 2006,
2010). A recent study by Lister et al. (2010) showed that
in addition to the SOD response, glutathione reductase,
glutathione peroxidase, and catalase are also altered by
UVR in sea urchin blastulae. In addition, the
Campanale et al. (2011) proteomic screen of cleavage-stage embryos revealed UV-induced changes in
a number of proteins that are hallmarks of oxidative
stress, including glutathione peroxidase, thioredoxin,
s-crystallin, and carbonic anhydrase (Table 1).
Proteomic analyses also identified cyclophilin D as
being UVR-responsive. This is an important mitochondrial chaperone that serves as an apoptosis sensor by
regulating the permeability of the transition pore complex, essential to detecting the redox state of the mitochondria, and recognizing intracellular Ca2þ flux
(Green and Reed 1998; Lin and Lechleiter 2002;
Baines et al. 2004). Therefore, cyclophilin D may be
sensing cellular redox states and UV-induced changes
in early sea urchin embryos after an assault by UV.
Protein turnover
Subunits of the major protein-degrading complex,
the proteasome, appear to be affected by UVR
Fig. 5 Response of a HSP70 to UVR. (A) Magnification of spots from composite proteomes (Fig. 3) that were identified as HSP70
isoform 3. (B) Comparisons of spot density shown in A indicate a UVR-dependent significant difference in spot density and a possible
shift in pI (suggestive of a PTM, ANOVA, P50.05). (C) One-dimensional immunoblots of total TX-soluble protein lysates sea urchin
embryos exposed to, or protected from, artificial UVR for 60 min from fertilization (QPanel UV340 lamps). (D) Immmunoblots of
2DE-separated proteins of UV-irradiated or protected embryos of Strongylocentrotus purpuratus exposed from 0 min to 60 min
post-fertilization. Samples were collected at 30 and 60 min. Gels in C and D were transferred to nitrocellulose, blocked, and probed
with Anti-HSP70 (Sigma #H5147). Binding was detected using GAM-HRP (BD Transduction Laboratories), and blots were imaged with
a Typhoon Trio Scanner (GE Healthcare).
674
N. L. Adams et al.
Fig. 6 Model of cellular responses in embryos exposed to UVR. Embryos protected from UVR progress through development while
maintaining cellular homeostasis and retaining a pool of defense-response proteins. During sub-lethal exposures to UVR, homeostasis is
temporarily lost and defensive-response proteins and pathways are activated to ameliorate UV-stress and protect against subsequent
exposure. These stress-response pathways may include sensors that are at the intersection of the repairing of molecular damage and
apoptosis. When these responses are no longer needed, homeostasis is restored and development resumes. However, after a lethal
dose of UVR, the defensive and stress-response pathways are not able to overcome the massive macromolecular damage and apoptotic
cascades are initiated, leading to death. See "Discussion" section.
(Campanale et al. 2011). More specifically, UVR
caused changes in the abundance of 26S proteasome
non-ATPase subunit, p44.5 subunit, and the proteasome 2 subunit in early sea urchin embryos. There
is a natural increase in proteasome activity during
activation of the egg and during the transition
from egg to embryo (Horner and Wolfner 2008),
but this appears heightened upon exposure to UV.
The identification of a large number of proteasome
subunits that showed changes in UV-treated embryos
(Table 1) indicates this entire complex may be involved in an important response to UVR.
Consistent with this, is the above-mentioned link
between ubiquitylation and stress responses (Vucic
et al. 2011). As a result of UV-stress, the proteasome
complex mediates the degradation of p53 in human
cells (Glockzin et al. 2003). Sea urchin embryos also
express p53 and it may play a role in the apoptotic
pathway activated after UV-stress during later stages
in development (Lesser et al. 2003). Not surprisingly,
inhibition of the proteasome arrests mitosis in sea
urchin embryos by delaying the degradation of
cyclin and also regulates exit from the S-phase of
the cell cycle (Kawahara et al. 2000). It seems reasonable that protein turnover would intimately control the molecular switches controlling the activity of
CSR pathways.
Translation
There is some evidence that UVR may be interfering
with the ability to translate maternally stored mRNA
in sea urchin embryos. Iordanov et al. (1998) suggested that part of the cellular response to UV-stress
in mammalian cells may be generated at the ribosome from damaged rRNA. Similarly, the proteomic
screen (Campanale et al. 2011) revealed multiple
proteins involved in translation that were responsive
to UVR treatment, including L10e/P0 (an acidic ribosomal protein), eIF2, and eIF4AII (proteins required for the initiation of translation), and
nucleolin, a phosphoprotein needed for the synthesis
and maturation of ribosomes. eIF2 kinases have
been shown to play a role in UV-induced apoptosis
in human cells, in part due to the phosphorylation of
eIF2 (Parker et al. 2006). Overexpression of nucleolin inhibits the translation of p53 whereas down regulation of nucleolin promotes the expression of p53
along with ribosomal subunits in mammals after
DNA damage (Takagi et al. 2005). Therefore, alteration of any of these proteins may affect translation
of all, or a subset of, mRNAs during first cleavage,
and may explain the observed delays in division.
Metabolism and transport
The proteomic screen also revealed UVR-responsiveness
in a suite of proteins that fall under the BioSystems
Proteomic responses of sea urchin embryos
metabolic pathway (Table 1). For example, levels of
mitochondrial isocitrate dehydrogenase 2 (NADPþ)
decreased in response to UVR. This enzyme plays a
role in recycling NADPþ within the mitochondria and
overexpression of it results in protection from
ROS-induced damage in mouse NIH3T3 cells (Jo et al.
2000). This observation suggests that sea urchin
embryos may have an altered capacity to sense and
deal with ROS that interfaces with metabolic pathways.
As another example, the activity of isocitrate dehydrogenase is regulated by glutathionylation during periods
of oxidative stress (Kil and Park 2005). To our knowledge, no studies have yet assessed global metabolic
responses to UVR in embryos or how this interfaces
with the other responsive pathways.
Conclusions and future directions
Proteome-wide and targeted studies have revealed
key pathways affected by UVR in early sea urchin
embryos (Table 1). How the components of these
pathways interact and how the pathways themselves
interface remain outstanding questions. We propose
that sea urchin zygotes are poised to cope with stressors, such as UVR, using maternally derived toolkits.
In Fig. 6, fertilized eggs subjected to UVR implement
defenses using proteins that are already in use as
mediators of early development. These include cell
cycle regulators such as the 14-3-3 proteins and
Cdc2, and modulators of metabolic homeostasis
and protein turnover. Many of these proteins are
involved in multiple systems; for example, the proteasome complex is crucial not only for protein turnover to regulate the cell cycle through cyclin
degradation, but also to clear mis-folded proteins
and remove maternal proteins no longer needed for
the egg to make the transition to an embryo. We
propose that UVR triggers these defensive proteins
(mostly through PTMs) as a first-line response. In
response to UVR, for example, proteasome activity
increases and cell cycle checkpoints initiate pauses in
response to DNA damage. However, if homeostasis
and the progression of the cell cycle are affected,
second-line CSRs deploy. Apoptotic sensors likely interface with these pathways. Collectively, one effect
of UVR is manifested in a delay of cell cycle progression. When the defensive and stress-response systems
are successful in responding to the damage (a
sub-lethal dose of UVR), homeostasis is restored
and progression of the cell cycle re-commences.
However, if defenses and CSRs are unsuccessful (a
675
lethal dose of UVR), cell-cycle halts, and death
ensues (Fig. 6). The same scenarios may apply to
embryos that are exposed to UVR at later stages in
development, with lethal doses resulting in aberrant
development (such as exogastrulation) or apoptosis,
but the defenses and CSRs may also be able to
employ robust transcriptional responses.
Functional studies to examine the proteins identified in proteomic studies are needed to resolve longstanding questions regarding the mechanisms by
which UV-stress alters cellular physiology and delays
development. Identification of specific PTMs of
UVR-responsive proteins is essential for understanding regulation and activity. While protein identification after 2DE is powerful for tracking individual
proteins, complementary, higher throughput proteomic technologies (c.f. Altelaar and Heck 2012) will
result in a more complete collection of proteins and
pathways that respond to UVR. Comprehensive and
unbiased assessments of UV-induced changes in proteomes can provide a deeper understanding of how
UVR affects overall cell physiology and the molecular
regulation of stress, thereby generating rich data sets
that can be mined for critical targets and regulators.
Furthermore, advancement of proteomic approaches
will help identify the proteins that are directly damaged by UVR, or are indirectly regulated after exposure to UVR. Importantly, these studies will begin to
explain how protein abundance and PTMs affect protein–protein interactions and thus how the various
pathways interact to coordinate a response to UVR.
Whether these same pathways and key proteins are
signature responses in later stages, or how they will
change with dose and wavelength of UVR also remain
to be determined. For example, a cleavage-stage
embryo may respond differently (with only maternally
derived proteins) than will later stages that can use
transcriptional mechanisms. It will be important to
evaluate whether maternal investment in protective
proteins to eggs influences these responses to UVR
across all stages and to perform field studies to validate laboratory results. The effects of UVR on multiple species at multiple stages will surely enrich our
understanding of responses to UVR and will allow the
mapping of individual proteins onto a comprehensive
set of UVR pathways. In turn, this will inform and
provide context for critical studies that look at the
synergistic effects of multiple stressors that are likely
to be encountered by marine organisms in a changing
environment.
676
Acknowledgments
We thank Lars Tomanek and the Division of
Comparative Physiology and Biochemistry of the
Society for Integrative and Comparative Biology for
inviting us to submit this manuscript. We thank
members of the Adams laboratory who helped
gather data for this manuscript. We apologize for
omitting many important primary references due to
constraints on space.
Funding
Funding for original data was provided by National
Science Foundation (NSF) Grant IBN-0417003
awarded to N.L.A.
References
Adams NL, Foltz KR. 2002. A molecular approach to understanding UV-induced mitotic delay in sea urchin embryos
Gulf of Mexico Sciences 19(2):16.
Adams NL, Shick JM. 1996. Mycosporine-like amino acids
provide protection against ultraviolet radiation in eggs of
the green sea urchin Strongylocentrotus droebachiensis.
Photochem Photobiol 64:149–58.
Adams NL, Shick JM. 2001. Mycosporine-like amino acids
prevent UVB-induced abnormalities during early development of the green sea urchin Strongylocentrotus droebachiensis. Mar Biol 138:267–80.
Agueli C, Geraci F, Giudice G, Chimenti L, Cascino D,
Sconzo G. 2001. A constitutive 70 kDa heat-shock protein
is localized on the fibres of spindles and asters at metaphase
in an ATP-dependent manner: a new chaperone role is
proposed. Biochem J 360:413–19.
Aiken CT, Kaake RM, Wang X, Huang L. 2011. Oxidative
stress-mediated regulation of proteasome complexes. Mol
Cell Proteomics 5:R110.006924-1–11.
Akimoto Y, Shiroya T. 1987a. Photoreversal of abnormal
morphogenesis in sea-urchin embryos caused by
UV-irradiation. Photochem Photobiol 45:403–6.
Akimoto Y, Shiroya T. 1987b. Changes in protein compliment
following UV-irradiation and their photoreversal in sea
urchin (Hemicentrotus pulcherrimus) embryos. Photochem
Photobiol 45:809–14.
Altelaar AM, Heck AJ. 2012. Trends in ultrasensitive proteomics.
Curr Opin Chem Biol 16:206–13.
Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H,
Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA,
Dorn GW, et al. 2004. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell
death. Nature 434:658–62.
Bancroft BA, Baker NJ, Blaustein AR. 2007. Effects of UVB on
marine and freshwater organisms: a synthesis through
meta-analysis. Ecol Lett 10:332–45.
N. L. Adams et al.
Blum H, Kauzmann E, Chapman G. 1954. Ultraviolet light
and the mitotic cycle in the sea urchin egg. J Gen Physiol
37:325–34.
Bonaventura R, Poma V, Costa C, Matranga V. 2005. UVB
radiation prevents skeleton growth and stimulates expression of stress markers in sea urchin embryos. Biochem
Biophys Res Commun 328:150–57.
Bonaventura R, Poma V, Russo R, Zito F, Matranga V. 2006.
Effects of UV-B radiation on development and hsp70 expression in sea urchin cleavage embryos. Mar Biol
149:79–86.
Borgne A, Meijer L. 1996. Sequential dephosphorylation of
p34cdc2 on Thr-14 and Tyr-15 at the prophase/metaphase
transition. J Biol Chem 271:27847–54.
Borra MT, Oniell FJ, Jackson MD, Marshall B, Verdin E,
Foltz KR, Denu JM. 2002. Conserved enzymatic production
and biological effect of O-Acetyl-ADP-ribose by silent information regulator 2-like NAD-dependent deacetylases. J
Biol Chem 277:12632–41.
Bothwell ML, Sherbot DM, Pollock CM. 1994. Ecosystem response to solar ultraviolet-B radiation: influence of
trophic-level interactions. Science 265:97–100.
Braconi D, Bernardini G, Santucci A. 2011. Linking protein
oxidation to environmental pollutants: redox proteomic
approaches. J Proteomics 74:2324–37.
Bradham CA, Foltz KR, Beane WS, Arnone MI, Rizzo F,
Coffman JA, Mushegian A, Goel M, Morales J,
Geneviere A-M, et al. 2006. The sea urchin kinome: a
first look. Dev Biol 300:180–93.
Brandhorst BP. 1976. Two-dimensional gel patterns of protein
synthesis before and after fertilization of sea urchin eggs.
Dev Biol 52:310–17.
Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, Basrur V,
Potapova O, Appella E, Fornace AJ. 2001. Initiation of a
G2/M checkpoint after ultraviolet radiation requires p38
kinase. Nature 411:102–7.
Byrum CA, Walton KD, Robertson AJ, Carbonneau S,
Thomason RT, Coffman JA, McClay DR. 2006. Protein tyrosine and serine–threonine phosphatases in the sea urchin,
Strongylocentrotus purpuratus: identification and potential
functions. Dev Biol 300(1):194–218.
Cadet J, Douki T, Gasparutto D, Ravanat JL. 2003. Oxidative
damage to DNA: formation, measurement and biochemical
features. Mutat Res 531:5–23.
Caldwell MM, Björn LO, Bornman JF, Kulandiavelu G,
Teramura AH, Tevini M. 1998. Effects of increased solar
ultraviolet radiation on terrestrial ecosystems. J Photochem
Photobiol B 46:40–52.
Campanale JP, Tomanek L, Adams NL. 2011. Exposure to
ultraviolet radiation causes proteomic changes in embryos
of the purple sea urchin, Strongylocentrotus purpuratus. J
Exp Mar Biol Ecol 397:106–20.
Carroll AK, Shick JM. 1996. Dietary accumulation of
UV-absorbing mycosporine-like amino acids, MAAs, by
the green sea urchin (Strongylocentrotus droebachiensis).
Mar Biol 124:561–69.
Ciechanover A. 1998. The ubiquitin-proteasome pathway: on
protein death and cell life. EMBO J 17:7151–60.
Proteomic responses of sea urchin embryos
Ciechanover A, Schwartz AL. 1998. The ubiquitin-proteasome
pathway: the complexity and myriad functions of proteins
death. Proc Natl Acad Sci USA 95:2727–30.
Cowen KJ, Storey KB. 2003. Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. J Exp Biol 206:1107–15.
Dahms HU, Lee JS. 2010. UV radiation in marine ectotherms:
molecular effects and responses. Aquat Toxicol 97:3–14.
Davidson EH. 2006a. The regulatory genome. Gene regulatory
networks in development and evolution. New York:
Academic Press. p. 289.
Davidson EH. 2006b. Special issue: the sea urchin genome.
Dev Biol 300:1.
Davidson EH, Cameron RA, Ransick A. 1998. Specification of
cell fate in the sea urchin embryo: summary and some
proposed mechanisms. Development 125:3269–90.
Denny MW, Shibata MF. 1989. Consequences of surf-zone
turbulence for settlement and external fertilization. Am
Nat 134:859–89.
Dheilly NM, Haynes PA, Bove U, Nair SV, Raftos DA. 2011.
Comparative proteomic analysis of a sea urchin
(Heliocidaris erythrogramma) antibacterial response revealed
the involvement of apextrin and calreticulin. J Invertebr
Pathol 106:223–29.
Dominguez R, Holmes KC. 2011. Actin structure and function. Annu Rev Biophys 40:169–86.
Downes A, Blunt TP. 1877. Researches on the effects of light
upon Bacteria and other organisms. Proc R Soc Lond
26:488–500.
Epel D. 2003. Protection of DNA during early development:
adaptations and evolutionary consequences. Evol Dev 5:83–8.
Feder ME, Hoffman GE. 1999. Heat-shock proteins, molecular chaparones and the stress response: evolutionary and
ecological physiology. Annu Rev Physiol 61:243–82.
Ferl RJ, Manak MS, Reyes MF. 2002. The 14-3-3s. Genome
Biol 3:3010.1–10.7.
Fernandez-Guerra A, Aze A, Morales J, Mulner-Lorillon O,
Cosson B, Cormier P, Bradham C, Adams NL,
Robertson AJ, Marzluff WF, et al. 2006. The genomic repertoire for cell cycle control and DNA metabolism in S.
purpuratus. Dev Biol 300:238–51.
Fu H, Subramanian RR, Masters SC. 2000. 14-3-3 Proteins:
structure, function, and regulation. Annu Rev Pharmacol
40:617–47.
Geer LY, Marchler-Bauer A, Geer RC, Han L, He J, He S,
Liu C, Shi W, Bryant SH. 2010. The NCBI BioSystems
database. Nucleic Acids Res. 38:D492–96.
Geraci F, Sconzo G. 2003. Constitutive HSP70 role in sea
urchin embryos during mitosis. Recent Res Devel Biophys
Biochem 3:327–32.
Geraci F, Agueli C, Giudice G, Sconzo G. 2003. Localization
of HSP70, Cdc2, and cyclin B in sea urchin oocytes in
non-stressed conditions. Biochem Biophys Res Commun
310:748–53.
Gething MJ, Sambrook J. 1992. Protein folding in the cell.
Nature 355:33–45.
Giese A. 1938. The effects of ultra-violet radiation of 2537A
upon cleavage of sea urchin eggs. Biol Bull 74:330–41.
677
Giese A. 1964. Studies on ultraviolet radiation action upon
animal cells. In: Giese A, editor. Photophysiology, Vol. II.
New York: Academic Press. p. 203–45.
Giudice G, Sconzo G, Roccheri MC. 1999. Studies on heat
shock proteins in sea urchin development. Dev Growth
Differ 41:375–80.
Gleason DF, Wellington GM. 1996. Variation in UVB sensitivity of planula larvae of the coral Agaricia agaricdes along
a depth gradient. Mar Biol 123:693–703.
Glockzin S, Ogi FX, Hengstermann A, Scheffner M,
Blattner C. 2003. Involvement of the DNA repair protein
hHR23 in p53 degradation. Mol Cell Biol 23:8960–69.
Goldstone JV, Hamdoun A, Cole BJ, Howard-Ashby M,
Nebert DW, Scally M, Dean M, Epel D, Hahn ME,
Stegean JJ. 2006. The chemical defensome: environmental
sensing and response genes in the Strongylocentrotus purpuratus genome. Dev Biol 300:366–84.
Gong ZY, Cserjesi P, Wessel GM. 1991. Structure and expression of the polyubiquitin gene in sea urchin embryos. Mol
Reprod Dev 28:111–8.
Grainger JL, von Brunn A, Winkler MM. 1986. Transient
synthesis of a specific set of proteins during the rapid cleavage phase of sea urchin development. Dev Biol 114:403–15.
Gravem S. 2009. Sex and microhabitat influence the allocation
of mycosporine-like amino acids to tissues in the purple sea
urchin, Strongylocentrotus purpuratus [Masters Thesis]. [San
Luis Obispo (CA)]: California Polytechnic State Univeristy.
Green DR, Reed JC. 1998. Mitochondria and apoptosis.
Science 281:1309–12.
Häder D, Sinha RP. 2005. Solar ultraviolet radiation-induced
DNA damage in aquatic organisms: potential environmental impact. Mut Res 571:221–33.
Häder DP, Helbling EW, Williamson CE, Worrest RC. 2011.
Effects of UV radiation on aquatic ecosystems and interactions with climate change. Photochem Photobiol Sci
10:242–60.
Hamdoun A, Epel D. 2007. Embryo stability and vulnerability
in an always changing world. Proc Natl Acad Sci USA
104:1745–50.
Helmbrecht K, Zeise E, Rensing L. 2000. Chaperones in cell
cycle regulation and mitogenic signal transduction: a
review. Cell Prolif 33:341–65.
Hoffman J, Lamare MD. 2004. Natural variation of carotenoids in the eggs and gonads of the echinoid genus,
Strongylocentrotus: implications for their role in ultraviolet
radiation photoprotection. J Exp Mar Biol Ecol 312:215–33.
Horner VL, Wolfner MF. 2008. Transitioning from egg to
embryo: triggers and mechanisms of egg activation. Dev
Dyn 237:527–44.
Isely N, Lamare M, Marshall C, Barker M. 2009. Expression of
the DNA repair enzyme, photolyase, in developmental tissues and larvae, and in response to ambient UV-R in the
Antarctic sea urchin Sterechinus neumayeri. Photochem
Photobiol 85:1168–76.
Iordanov MS, Pribnow D, Magun JL, Dinh TH, Pearson JA,
Magun BE. 1998. Ultraviolet radiation triggers the ribotoxic
stress response in mammalian cells. J Biol Chem
273:15794–803.
678
Janke C, Bulinski JC. 2011. Post-translational regulation of
the microtubule cytoskeleton: mechanisms and function.
Nat Rev Mol Cell Biol 12:773–86.
Jo SH, Son MK, Koh HJ, Lee SM, Song IH, Kim YO,
Lee YS, Jeong KS, Kim WB, Park JW, et al. 2000.
Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial
NADPþ-dependent isocitrate dehydrogenase. J Biol
Chem 276:16168–76.
Kalume DE, Molina H, Pandey A. 2003. Tackling the phosphoproteome: tools and strategies. Curr Opin Chem Biol
7:64–9.
Karentz D. 1994. Ultraviolet tolerance mechanisms in
Antarctic marine organisms. In: Weiler CS, Penhale PA,
editors. Ultraviolet radiation and biological research in
Antarctica. Antarctic Research Series V 62. Washington,
DC: American Geophysical Union. p. 93–110.
Kawahara H, Phillipova R, Yokosawa H, Patel R, Tanaka K,
Whitaker M. 2000. Inhibiting proteasome activity causes
overreplication of DNA and blocks entry into mitosis in
sea urchin embryos. J Cell Sci 113:2659–70.
Kiang JG, Tsokos GC. 1998. Heat shock protein 70 kDa: molecular biology, biochemistry and physiology. Parmacol
Ther 80:183–201.
Kil IS, Park JW. 2005. Regulation of mitochondrial
NADPþ-dependent isocitrate dehydrogenase activity by
glutathionylation. J Biol Chem 280:10846–54.
Kulms D, Dübmann H, Pöppelmann B, Ständer S, Schwarz A,
Schwarz T. 2002. Apoptosis induced by disruption of the
actin cytoskeleton is mediated via activation of CD95 (Fas/
APO-1). Cell Death Diff 9: 598–608.
Kültz D. 2005. Molecular and evolutionary basis of the cellular stress response. Annu Rev Physiol 67:225–57.
Lamare MD, Burritt D, Lister K. 2011. Ultraviolet radiation
and echinoderms: past, present and future perspectives.
In: Lesser MP, editor. Advances in Marine Biology, Vol.
59. 2nd ed. Plymouth, UK: Elsevier Ltd. p. 145–87.
Lamare MD, Barker MF, Lesser MP. 2007. In situ rates of
DNA damage and abnormal development in Antarctic and
non-Antarctic sea urchin embryos. Aqua Biol 1:21–32.
Lesser MP. 2006. Oxidative stress in marine environments:
biochemistry and physiologicalecology. Ann Rev Physiol
68:253–78.
Lesser MP. 2010. Depth-dependent effects of ultraviolet
radiation on survivorship, oxidative stress and DNA
damage in sea urchin (Strongylocentrotus droebachiensis)
embryos from the Gulf of Maine. Photochem Photobiol
8:382–88.
Lesser M, Barry T, Lamare M, Barker M. 2006. Biological
weighting functions for DNA damage in sea urchin
embryos exposed to ultraviolet radiation. J Exp Mar Bio
Ecol 328:10–21.
Lesser M, Kruse V, Barry T. 2003. Exposure to ultraviolet
radiation causes apoptosis in developing sea urchin
embryos. J Exp Biol 206:4097–103.
Levee MG, Dabrowska MI, Lelli JL, Hinshaw DB. 1996. Actin
polymerization and depolymerization during apoptosis in
HL-60 cells. Am J Cell Physiol 271:C1981–92.
N. L. Adams et al.
Lin D, Lechleiter JD. 2002. Mitochondrial targeted cyclophilin
D protects cells from cell death by peptidyl prolyl isomerization. J Biol Chem 277:31134–41.
Lister KN, Lamare MD, Burritt DJ. 2010. Oxidative damage in
response to natural levels of UV-B radiation in larvae of the
tropical sea urchin Tripneustes gratilla. Photochem
Photobiol 86:1091–98.
Liu Q, Guntuku S, Cui X, Matsuoka S, Cortez D. 2000. Chk1
is an essential kinase that is regulated by Atr and required
for the G2/M DNA damage checkpoint. Genes Dev
14:1448–59.
Madronich S, Mckenzie R, Bjorn L, Caldwell M. 1998.
Changes in biologically active ultraviolet radiation reaching
the Earth’s surface. In: der Leun J, Tang X, Tevini M,
editors. Environmental effects of ozone depletion: 1998
assessment. New York: Environment Programme Report,
United Nations. p. 5–19.
Majmundar AJ, Wong WJ, Simon MC. 2010.
Hypoxia-inducible factors and the response to hypoxic
stress. Mol Cell 40:294–309.
Mann M, Jensen O. 2003. Proteomic analysis of
post-translational modifications. Nat Biotech 21:255–61.
Mann K, Wilt FH, Poustka AJ. 2010. Proteomic analysis of
sea urchin Strongylocentrotus purpuratus spicule matrix.
Proteome Sci 17:8–33.
McKenzie RL, Aucamp PJ, Bais AF, Björn LO, Ilyas M. 2007.
Changes in biologically-active ultraviolet radiation reaching
the Earth’s surface. Photochem Photobiol Sci 6:218–31.
McKenzie RL, Bijorn L, Bais A, Islyasd M. 2003. Changes in
biologically active ultraviolet radiation reaching the Earth’s
surface. Photochem Photobiol Sci 2:5–15.
Miller B, Emlet R. 1997. Influence of nearshore hydrodynamics on larval abundance and settlement of sea urchins
Strongylocentrotus franciscanus and S. purpuratus in the
Oregon upwelling zone. Mar Ecol Prog Ser 148:83–94.
Mostajir B, Demers S, de More SJ, Bukata RP, Jerome JH.
2000. Implications of UV radiation for the food web structure and consequences on the carbon flow. In: de Mora S,
Demers S, Vernet M, editors. The Effects of UV Radiation
in the Marine Environment. Cambridge: Cambridge
University Press. p. 310–20.
Murray A, Kirschner M. 1989. Cyclin synthesis drives the
early embryonic cell cycle. Nature 339:275–80.
Niu P, Liu L, Gong Z, Tan H, Wang F, Yuan J, Feng Y,
Wei Q, Tanguay RM, Wu T. 2006. Overexpressed heat
shock protein 70 protects cells against DNA damage
caused by ultraviolet C in a dose-dependent manner. Cell
Stress and Chaparones 11:162–9.
Parker SH, Parker TA, George KS, Wu S. 2006. The roles of
translation initiation regulation in ultraviolet light-induced
apoptosis. Mol Cell Biochem 293:173–81.
Pechenik JA. 1987. Environmental influences on larval survival and development. In: Giese AC, Pearse JS,
Pearse VB, editors. Reproduction of marine invertebrates,
Vol. IX. Cambridge: Blackwell Scientific Publications and
Boxwood Press. p. 511–608.
Pennington JT, Emlet RB. 1986. Ontogenetic and diel vertical
migration of a planktonic echinoid larvae, Dendraster
Proteomic responses of sea urchin embryos
excentricus (Eschscholtz): occurrence, causes, and probable
consequences. J Exp Mar Biol Ecol 104:69–95.
Polevoda B, Sherman F. 2002. The diversity of acetylated proteins. Genome Biol 3:1–6.
Przeslawski R, Davis AR, Benkendorff K. 2005. Synergistic
effects associated with climate change and the development of rocky shore mollusks. Global Change Biol
11:515–22.
Robinson DN, Spudich JA. 2000. Towards a molecular understanding of cytokinesis. Trends Cell Biol 10:228–37.
Roux MM, Radeke M, Goel M, Mushegian A, Foltz KR. 2008.
2DE identification of proteins exhibiting turnover and
phosphorylation dynamics during sea urchin egg activation.
Dev Biol 313:630–47.
Roux MM, Townley IK, Raish M, Reade A, Bradham CA,
Humphreys G, Gunaratne HJ, Killian KE, Moy G, Su Y,
et al. 2006. A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation. Dev
Biol 300:416–33.
Russo R, Zito F, Costa C, Bonaventura R, Matranga V. 2010.
Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B. Cell Stress
Chaperones 15:1–9.
Rustad R. 1960. Changes in the sensitivity to
ultraviolet-induced mitotic delay during the cell division
cycle of the sea urchin egg. Exp Cell Res 21:596–602.
Rustad R. 1964. U.V.-induced mitotic delay in the sea urchin
egg. Photochem Photobiol 3:529–38.
Rustad R. 1971. Radiation response during the mitotic cycle
of the sea urchin egg. In: Cameron, Padilla G,
Zimmerman A, editors. Developmental aspects of the cell
cycle. New York: Academic Press. p. 127–59.
Samanta MP, Tongprasit W, Istrail S, Cameron RA, Tu Q,
Davidson EH, Stolc V. 2006. The transcriptome of the sea
urchin embryo. Science 314:960–62.
Sconzo G, Palla F, Agueli C, Spinelli G, Giudice G, Cascino D,
Geraci F. 1999. Constitutive hsp70 is essential to mitosis
during early cleavage of Paracentrotus lividus embryos: the
blockage of constitutive hsp70 impairs mitosis. Biochem
Biophys Res Commun 260:143–9.
Sea Urchin Genome Sequencing Consortium. 2006. The
genome of the sea urchin Strongylocentrotus purpuratus.
Science 314:941–52.
Shapiro BM. 1991. The control of oxidant stress at fertilization. Science 252:533–36.
Sinha RP, Häder DP. 2002. UV-induced DNA damage and
repair: a review. Photchem Photobiol Sci 1:225–36.
Smith RC, Baker K. 1979. Penetration of UV-B and biologically effective dose-rates in natural waters. Photochem
Photobiol 29:311–23.
Sodergren E, Shen Y, Song X, Zhang L, Gibbs RA,
Weinstock GM. 2006. Shedding light on Aristotle’s
Lantern. Dev Biol 300:2–8.
Sun HQ, Yamamoto M, Mejullano M, Yin HL. 1999.
Gelsolin, a multifunctional actin regulatory protein. J Biol
Chem 274:33179–82.
Takagi M, Absalon MJ, McLure KG, Kastan MB. 2005.
Regulation of p53 translation and induction after DNA
679
damage by ribosomal protein and nucleolin. Cell
123:49–63.
Tanaka K. 2010. Multiple functions of the S-phase checkpoint
mediator. Biosci Biotechnol Biochem 74:2367–73.
Tevini M. 1993. Molecular biological effects of ultraviolet radiation. In: Tevini M, editor. UV-B Radiation and ozone
depletion: effects on humans, animals, plants, microorganisms, and materials. Baca Raton, FL: Lewis Publishers.
p. 1–16.
Tomanek L. 2010. Variation in the heat shock response and
its implication for predicting the effect of global climate
change on species’ biogeographical distribution ranges
and metabolic costs. J Exp Biol 213:971–79.
Tomanek L. 2011. Environmental proteomics: changes in the
proteome of marine organisms in response to environmental stress, pollutants, infection, symbiosis and development.
Annual Rev Mar Sci 3.3:14.1–27.
Tomanek L, Zuzow MJ. 2010. The proteomic response of the
mussel congeners Mytilus galloprovincialis and M. trossulus
to acute heat stress: implications for thermal tolerance
limits and metabolic costs of thermal stress. J Exp Biol
213:3559–74.
Tyrrell L. 1991. UV-A 320-380 nm Radiation as an oxidative
stress. In: Sies H, editor. Oxidative stress: oxidants and
antioxidants. San Diego, CA: Academic Press. p. 57–83.
United Nations Environment Programme, Environmental
Effects Assessment Panel. 2006. Environmental effects of
ozone depletion and its interactions with climate
change: Progress report, 2005. Photochem Photobiol Sci
5:13–24.
Van Heusden GP. 2009. 14-3-3 proteins: insights from
genome-wide studies in yeast. Genomics 94:287–93.
Vega RL, Epel D. 2004. Stress-induced apoptosis in sea urchin
embryogenesis. Mar Environ Res 58:799–802.
Verma R, Chen S, Feldman S, Schieltz D, Yates J, Dohmen J,
Deshaies RJ. 2000. Proteasomal proteomics: identification
of nucleotide-sensitive proteasome-interacting proteins by
mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 11:3425–39.
Vincent WF, Neale PJ. 2000. Mechanism of UV damage to
aquatic organisms. In: deMora S, Demers S, Vernet M,
editors. The effects of UV radiation in the marine environment. Cambridge: Cambridge University Press.
p. 149–76.
Voronina E, Wessel GM. 2001. Apoptosis in sea urchin oocytes, eggs and sea urchin embryos. Mol Repro Dev
60:553–61.
Vucic D, Dixit VM, Wertz IE. 2011. Ubiquitylation in apoptosis: a post-translational modification at the edge of life
and death. Nat Rev Mol Cell Biol 12:439–52.
Westerheide SD, Anckar J, Stevens SM Jr, Sistonen L,
Morimoto RI. 2009. Stress-inducible regulation of heat
shock factor 1 by the deacetylase SIRT1. Science
323:1063–6.
Whitaker M. 2006. Calcium at fertilization and in early development. Physiol Rev 86:25–88.
Whitehead RF, de Mora S, Demers S, Gosselin M, Monfort P,
Mostajir B. 2000. Interactions of ultraviolet-B radiation,
680
mixing and biological activity on photobleaching of natural
chromophoric dissolved organic material: a mesocosm
study. Limnol Oceanogr 45:278–91.
Wong J, Créton R, Wessel G. 2004. The oxidative burst at
fertilization is dependent upon activation of the dual oxidase Udx1. Dev Cell 7:801–14.
World Meteorological Organization. 2007. Scientific
Assessment of Ozone Depletion 2006-SummaryGlobal
Ozone Research Monitoring Project-Report 50.
Worrest R. 1982. Review of literature concerning the impact
of UV-B radiation upon marine organisms. NATO Conf
Ser 7:429–57.
N. L. Adams et al.
Zamansky GB, Chou IN. 1987. Environmental wavelengths of
ultraviolet light induce cytoskeletal damage. J Invest
Dermatol 89:603–6.
Zepp RG, Erickson DJ III, Paul ND, Sulzberger B. 2011.
Effects of solar UV radiation and climate change on biogeochemical cycling: interactions and feedbacks. Photochem
Photobiol Sci 10:261–79.