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Kalderon, D., Roberts, B.L., Richardson, W.D. & Smith, A.E. A short amino acid sequence able to specify nuclear location. Cell 39, 499-509

  • January 1985
  • Cell 39(3 Pt 2):499-509
DOI:10.1016/0092-8674(84)90457-4
Authors:
Daniel Kalderon at Columbia University
Daniel Kalderon
Bruce L. Roberts at Sanofi
Bruce L. Roberts
William David Richardson at University College London
William David Richardson
Alan E. Smith
Alan E. Smith
Alan E. Smith
  • This person is not on ResearchGate, or hasn't claimed this research yet.
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Abstract and Figures

A short sequence of amino acids including Lys-128 is required for the normal nuclear accumulation of wild-type and deleted forms of SV40 large T antigen. A cytoplasmic large T mutant that lacks sequences from around Lys-128 localizes to the nucleus if the missing sequence is attached to its amino terminus. The implication that the sequence element around Lys-128 acts as an autonomous signal capable of specifying nuclear location was tested directly by transferring it to the amino termini of beta-galactosidase and of pyruvate kinase, normally a cytoplasmic protein. Sequences that included the putative signal induced each of the fusion proteins to accumulate completely in the nucleus but had no discernible effect when Lys-128 was replaced by Thr. By reducing the size of the transposed sequence we conclude that Pro-Lys-Lys-Lys-Arg-Lys-Val can act as a nuclear location signal. The sequence may represent a prototype of similar sequences in other nuclear proteins.
Subcellular Locations of Large T Variants Containing Two Copres. or One Relocated Copy of a Short Sequence around Residue 128 Vero cells were microinjected with plasmrd DNAs encoding the variant large T molecules shown in Figure 3. Following microinjection, cells were fixed and starned using monoclonals PAb423 (a to d) or PAb419 (e and f) (Harlow et al.. 1981) as first antibody. The second antibody was rhodamine-labeled rabbit anti-mouse IgG (Fab; fragment). Micrographs a to d represent large T variants containing two consecutive copies of the ammo acid seqeunce from 127 to 135 wrth either Lys or Thr at positron 128, as follows (a) Lys-128 in both copies, L27-L7; (b) Thr-128 in both copies, dlO/L27-dlO/L7; (c) Lys-128 followed by Thr-128, L27-dlO/L7; (d) Thr-128 followed by Lys-128, dlO/L27-L7. Mrcrographs (e) and (f) show the localization of large T derivatives in which amino acids 127 to 147 including either Lys or Thr at posrtion 128 have been added to the amino terminus of the cytoplasmic mutant Sl l-S33 as follows: (e) Lys at posrtron 128, L7RH/Sl l-S33; (f) Thr at posrtron 128, dlO/L7RH/Sll-S33.
Subcellular Locations of Large T Variants Containing Two Copres. or One Relocated Copy of a Short Sequence around Residue 128 Vero cells were microinjected with plasmrd DNAs encoding the variant large T molecules shown in Figure 3. Following microinjection, cells were fixed and starned using monoclonals PAb423 (a to d) or PAb419 (e and f) (Harlow et al.. 1981) as first antibody. The second antibody was rhodamine-labeled rabbit anti-mouse IgG (Fab; fragment). Micrographs a to d represent large T variants containing two consecutive copies of the ammo acid seqeunce from 127 to 135 wrth either Lys or Thr at positron 128, as follows (a) Lys-128 in both copies, L27-L7; (b) Thr-128 in both copies, dlO/L27-dlO/L7; (c) Lys-128 followed by Thr-128, L27-dlO/L7; (d) Thr-128 followed by Lys-128, dlO/L27-L7. Mrcrographs (e) and (f) show the localization of large T derivatives in which amino acids 127 to 147 including either Lys or Thr at posrtion 128 have been added to the amino terminus of the cytoplasmic mutant Sl l-S33 as follows: (e) Lys at posrtron 128, L7RH/Sl l-S33; (f) Thr at posrtron 128, dlO/L7RH/Sll-S33.
… 
Subcellular Location of @-Galactosidase and Pyruvate Kinase Fusion Proteins Vero cells were microinjected with plasmid DNAs encoding fusion proteins illustrated in Figure 5 (P-galactosidase fusions) and Figures 7 and 8 (pyruvate kinase fusions). Eighteen hours later the cells were fixed and the fusion proteins were visualized by immunofluorescence microscopy. Micrographs (a) to (d) represent fl-galactosidase fusions, visualized with a mixture of monoclonals directed against @-galactosidase followed by rhodamine-conjugated rabbit antimouse IgG, as follows. (a) L27-BGAL, (b) dlO/L27-BGAL, (c) L7RH-BGAL, (d) dlO/L7RH-BGAL (see Figure 5). Micrographs (e) to (I) show pyruvate kinase fusions stained with a serum raised in rabbits against purified chicken muscle pyruvate kinase, and counter-stained with fluorescein-labeled goat anti-rabbit serum, as follows. (e) L27-PK, (f) dlO/L27-PK, (g) XR6PK, (h) XRdlO/L27-PK, (i) XRBO-PK, (i) XR22-PK, (k) m30-PJ-B, (1) xm30-PK (see Figures 7 and 8).
Subcellular Location of @-Galactosidase and Pyruvate Kinase Fusion Proteins Vero cells were microinjected with plasmid DNAs encoding fusion proteins illustrated in Figure 5 (P-galactosidase fusions) and Figures 7 and 8 (pyruvate kinase fusions). Eighteen hours later the cells were fixed and the fusion proteins were visualized by immunofluorescence microscopy. Micrographs (a) to (d) represent fl-galactosidase fusions, visualized with a mixture of monoclonals directed against @-galactosidase followed by rhodamine-conjugated rabbit antimouse IgG, as follows. (a) L27-BGAL, (b) dlO/L27-BGAL, (c) L7RH-BGAL, (d) dlO/L7RH-BGAL (see Figure 5). Micrographs (e) to (I) show pyruvate kinase fusions stained with a serum raised in rabbits against purified chicken muscle pyruvate kinase, and counter-stained with fluorescein-labeled goat anti-rabbit serum, as follows. (e) L27-PK, (f) dlO/L27-PK, (g) XR6PK, (h) XRdlO/L27-PK, (i) XRBO-PK, (i) XR22-PK, (k) m30-PJ-B, (1) xm30-PK (see Figures 7 and 8).
… 
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Content uploaded by William David Richardson
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;gJuclear Location Signal
either wholly or in part to the nuclear accumulation of all
the deleted and truncated forms of large T examined.
Because it operates in the context of so many variant
structures, this result suggests that the sequence around
Lys-128 does not rely on an interaction with other parts of
the molecule to ensure the nuclear location of large T.
Nuclear Accumulation of Large T Is Positively
Promoted by Sequences Including Lys-128
It has been assumed above that Lys-128 forms part of a
positive signal that promotes nuclear localization, rather
than Thr-128 inducing cytoplasmic retention of large T.
The latter possibility seemed unlikely because a number
of deletion mutants that remove Lys-128 and adjacent
sequences also share the cytoplasmic phenotype (Kalde-
ron et al., 1984). Nevertheless, this possibility was exam-
ined directly by constructing large T variants that contained
two successive copies of a short sequence (amino acids
127 to 135) around residue 128 with either Thr or Lys at
this position (Figure 3). In every case where a wild-type
sequence (including Lys at 128) was present, the variant
protein localized exclusively to the nucleus, irrespective of
whether this sequence was preceded or followed by se-
quences derived from d10 large T (Figures 3, 4a, 4c, 4d).
The same result was observed when the size of the
duplication was increased to 21 amino acids (127 to 147)
although in this case only one of the two orientations of
wild-type relative to mutant sequence was examined (data
not shown). Thus the presence of Thr-128 in this position
relative to the normal amino or carboxy terminus of large
T does not necessarily induce cytoplasmic retention,
whereas nuclear accumulation is promoted in every case
when Lys occupies position 128.
The unimpaired nuclear accumulation of molecules with
small duplications (Figure 4a, 4c, 4d) provides further
evidence, similar to that derived from analysis of deletion
mutants, that the function of the region around Lys-128 is
not critically dependent on its position relative to the rest
of the molecule.
Relocation of Putative Nuclear Location Signal to
the Amino Terminus of Large T
The insertion/deletion mutant Sl 1 S33 lacks native large T
amino acid residues between 127 and 132 and fails to
localize to the nucleus (Kalderon et al., 1984). We sought
to restore a nuclear location to this protein by introducing
at the amino terminus a short sequence of amino acids
from around Lys-128 in wild-type large T (Figure 3 and
section 4 of Experimental Procedures). Again, an analo-
gous construction was made in which Lys-128 was re-
placed by a Thr residue. The subcellular distribution of
these proteins was examined following microinjection of
plasmid DNA into Vero cells by immunofluorescent staining
using a monoclonal antibody directed against the amino
terminus of large T PAb419 (Harlow et al., 1981). Virtually
all of the chimeric protein with an amino-terminal basic
tract including Lys-128 was seen in the nucleus, whereas
the Thr-containing large T variant was entirely cytoplasmic
Laree-T E.COill H,“dlll s11-553
rerlduer ,tn*er
t-4 Llrge-7 re.ld”er 127-s47 ,>n*er ,er,d”er ,-End
m--H-
o ’
MO*YFPNSI.LIRXYEDPKOFPSELLSFLSPSF*X9
b%f T
Figure 3. Structure and Subcellular Location of Large T Variants Containing
Two Copies or One Relocated Copy of a Short Sequence around Residue
126
The construction of plasmids encodrng large T variants in which sequences
around Lys-128 were (a) duplrcated or (b) transposed from their normal
locatron to the ammo terminus IS described rn sections 1, 3, and 4 of
Experrmental Procedures. The ammo acid sequence followrng Met-36 of
L7RH/SI I-533 is identical with that following Met-l of the lanker insertton/
deletion mutant, Sl l-S33 (whrch lacks residues 127 to 132). The location
of vanant proteins was assessed followrng microinjection of plasmid DNA
Into Vero ceils by Indirect immunofluorescence microscopy (see Figure 4).
(Figures 4e and 4f). Thus a short sequence around Lys-
128 is able to confer a nuclear location to large T even
when moved by over a hundred amino acids within the
primary structure.
The tolerance of this sequence to transposition suggests
that it constitutes an independent structural element but
does not rule out the alternative possibility that its function
depends on an interaction with other regions of the large
T molecule. The hypothesis that the sequence acts as an
autonomous nuclear localization signal was therefore
tested more rigorously by ascertaining whether it is suffi-
cient to promote the nuclear accumulation of proteins that
are normally found in the cytoplasm. Plasmids encoding
chimeric proteins were therefore constructed in which the
putative nuclear location signal was close to a terminus of
the test protein where, it could be argued, there is most
chance of maintaining a structure that is independent of
the rest of the molecule.
Localization of Chimeric @-Galactosidase Proteins
P-gaiactosidase, encoded by the lac Z gene of, E. coli,
was initially selected as a suitable test protein for several
reasons. As a bacterial protein it would not be expected
to include specific signals that facilitate transport across
the nuclear envelope, although it may have an affinity for
Cdl
502
Figure 4. Subcellular Locations of Large T Variants Containing Two Copres. or One Relocated Copy of a Short Sequence around Residue 128
Vero cells were microinjected with plasmrd DNAs encoding the variant large T molecules shown in Figure 3. Following microinjection, cells were fixed and
starned using monoclonals PAb423 (a to d) or PAb419 (e and f) (Harlow et al.. 1981) as first antibody. The second antibody was rhodamine-labeled rabbit
anti-mouse IgG (Fab; fragment). Micrographs a to d represent large T variants containing two consecutive copies of the ammo acid seqeunce from 127 to
135 wrth either Lys or Thr at positron 128, as follows (a) Lys-128 in both copies, L27-L7; (b) Thr-128 in both copies, dlO/L27-dlO/L7; (c) Lys-128 followed by
Thr-128, L27-dlO/L7; (d) Thr-128 followed by Lys-128, dlO/L27-L7. Mrcrographs (e) and (f) show the localization of large T derivatives in which amino acids
127 to 147 including either Lys or Thr at posrtion 128 have been added to the amino terminus of the cytoplasmic mutant Sl l-S33 as follows: (e) Lys at
posrtron 128, L7RH/Sl l-S33; (f) Thr at posrtron 128, dlO/L7RH/Sll-S33.
nuclear components common to procaryotic and eucar-
yotic cells. The large size of the monomeric protein (M,
116,000; Fowler and Zabin, 1977) and its propensity to
oligomerize (Wallenfels and Weil, 1972) might be expected
to preclude rapid entry to the nucleus in the absence of a
specific transport mechanism. Furthermore, of the P-galac-
tosidase fusion proteins that have previously been con-
structed, many retain enzyme activity (Casadaban et al.,
1983) indicating that additional amino acid sequences can
be accommodated at either the amino or carboxy terminus
of the protein without gross effect on its native structure.
Finally, the same approach has been used previously to
identify sequences that localize @-galactosidase to the
nucleus in yeast (Hall et al., 1984).
Initially, four types of plasmid encoding P-galactosidase
fusion proteins were made (section 5 in Experimental
Procedures). All contained the same promoter, translation
initiation, and polyadenylation signals (all derived from the
SV40 early region) and the same /I-galactosidase coding
sequence (Figure 5). They differed only in the number of
SV40 large T amino acids that preceded the fusion junction
with fi-galactosidase. The subcellular location of each of
the fusion proteins was examined, following microinjection
of the appropriate plasmid DNA into Vero cells, by indirect
immunofluorescence using a cocktail of four monoclonal
antibodies raised against E. coli fi-galactosidase (a gift
from Dr. J. Partaledis).
Surprisingly, the hybrid protein that included only four
amino acids from the amino terminus of SV40 large T
(SRLSO-BGAL, Figure 5) was seen at approximately equal
intensities in both nuclear and cytoplasmic compartments.
We obtained the same result with a hybrid protein, pCH1 IO
(Hall et al., 1983) that included 40 amino acids encoded
by the E. coli gpt gene and 28 by the fro S gene at the
amino terminus of fi-galactosidase. Similar results have
been obtained by others for a series of amino- and car-
boxy-terminal P-galactosidase fusion proteins (D. P. Lane,
personal communication). Thus, despite its large size and
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Cell
504
l3-galactosidase pyruvate kinase
Figure 6. Subcellular Location of @-Galactosidase and Pyruvate Kinase Fusion Proteins
Vero cells were microinjected with plasmid DNAs encoding fusion proteins illustrated in Figure 5 (P-galactosidase fusions) and Figures 7 and 8 (pyruvate
kinase fusions). Eighteen hours later the cells were fixed and the fusion proteins were visualized by immunofluorescence microscopy. Micrographs (a) to (d)
represent fl-galactosidase fusions, visualized with a mixture of monoclonals directed against @-galactosidase followed by rhodamine-conjugated rabbit anti-
mouse IgG, as follows. (a) L27-BGAL, (b) dlO/L27-BGAL, (c) L7RH-BGAL, (d) dlO/L7RH-BGAL (see Figure 5). Micrographs (e) to (I) show pyruvate kinase
fusions stained with a serum raised in rabbits against purified chicken muscle pyruvate kinase, and counter-stained with fluorescein-labeled goat anti-rabbit
serum, as follows. (e) L27-PK, (f) dlO/L27-PK, (g) XR6PK, (h) XRdlO/L27-PK, (i) XRBO-PK, (i) XR22-PK, (k) m30-PJ-B, (1) xm30-PK (see Figures 7 and 8).
acid sequence that suffices to direct pyruvate kinase to
the nucleus. signals at their amino termini was, in principle, the same
as that used for P-galactosidase. End points, defined by
restriction enzyme cleavage sites, were created in each of
Large T Amino Acid Sequences Sufficient for the three possible reading frames both following the amino-
Nuclear Localization of Pyruvate Kinase terminal five residues of large T and preceding residue 17
The method used to construct plasmids encoding pyruvate of pyruvate kinase. Thus any small fragment of known
kinase fusion proteins with short putative nuclear location nucleotide sequence could be inserted between appropri-
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Peptide-Mediated Targeted Delivery of Aloe-Emodin as Anticancer Drug
Article
Full-text available
Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs' cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.
Understanding the role of mechanics in nucleocytoplasmic transport
Article
  • Jun 2022
Cell nuclei are submitted to mechanical forces, which in turn affect nuclear and cell functions. Recent evidence shows that a crucial mechanically regulated nuclear function is nucleocytoplasmic transport, mediated by nuclear pore complexes (NPCs). Mechanical regulation occurs at two levels: first, by force application to the nucleus, which increases NPC permeability likely through NPC stretch. Second, by the mechanical properties of the transported proteins themselves, as mechanically labile proteins translocate through NPCs faster than mechanically stiff ones. In this perspective, we discuss this evidence and the associated mechanisms by which mechanics can regulate the nucleo-cytoplasmic partitioning of proteins. Finally, we analyze how mechanical regulation of nucleocytoplasmic transport can provide a systematic approach to the study of mechanobiology and open new avenues both in fundamental and applied research.
Regulating Phase Transition in Neurodegenerative Diseases by Nuclear Import Receptors
Article
Full-text available
  • Jul 2022
RNA-binding proteins (RBPs) with a low-complexity prion-like domain (PLD) can undergo aberrant phase transitions and have been implicated in neurodegenerative diseases such as ALS and FTD. Several nuclear RBPs mislocalize to cytoplasmic inclusions in disease conditions. Impairment in nucleocytoplasmic transport is another major event observed in ageing and in neurodegenerative disorders. Nuclear import receptors (NIRs) regulate the nucleocytoplasmic transport of different RBPs bearing a nuclear localization signal by restoring their nuclear localization. NIRs can also specifically dissolve or prevent the aggregation and liquid–liquid phase separation of wild-type or disease-linked mutant RBPs, due to their chaperoning activity. This review focuses on the LLPS of intrinsically disordered proteins and the role of NIRs in regulating LLPS in neurodegeneration. This review also discusses the implication of NIRs as therapeutic agents in neurogenerative diseases.
Peptides to Overcome the Limitations of Current Anticancer and Antimicrobial Nanotherapies
Article
Full-text available
  • Jun 2022
Biomedical research devotes a huge effort to the development of efficient non-viral nanovectors (NV) to improve the effectiveness of standard therapies. NVs should be stable, sustainable and biocompatible and enable controlled and targeted delivery of drugs. With the aim to foster the advancements of such devices, this review reports some recent results applicable to treat two types of pathologies, cancer and microbial infections, aiming to provide guidance in the overall design of personalized nanomedicines and highlight the key role played by peptides in this field. Additionally, future challenges and potential perspectives are illustrated, in the hope of accelerating the translational advances of nanomedicine
The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains
Article
Full-text available
Calcium (Ca2+) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca2+ concentration ([Ca2+]i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca2+]i are ideally suited to modulate Ca2+-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca2+, the introduction and the ongoing development of genetically encoded Ca2+ indicators (GECI) opened a new era to study Ca2+-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca2+ signals at the (sub)-cellular level with pronounced temporal resolution.
Mechanical force application to the nucleus regulates nucleocytoplasmic transport
Article
Full-text available
Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Such forces can lead to the nuclear translocation of proteins, but whether force controls nucleocytoplasmic transport, and how, remains unknown. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than for facilitated diffusion. Owing to this differential effect, force leads to the translocation of cargoes into or out of the nucleus within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism and engineered exogenously by introducing appropriate nuclear localization signals. Our work unveils a mechanism of mechanically induced signalling, probably operating in parallel with others, with potential applicability across signalling pathways.
A Secreted Lignin Peroxidase Required for Fungal Growth and Virulence and Related to Plant Immune Response
Article
Full-text available
Botryosphaeria spp. are important phytopathogenic fungi that infect a wide range of woody plants, resulting in big losses worldwide each year. However, their pathogenetic mechanisms and the related virulence factors are rarely addressed. In this study, seven lignin peroxidase (LiP) paralogs were detected in Botryosphaeria kuwatsukai, named BkLiP1 to BkLiP7, respectively, while only BkLiP1 was identified as responsible for the vegetative growth and virulence of B. kuwatsukai as assessed in combination with knock-out, complementation, and overexpression approaches. Moreover, BkLiP1, with the aid of a signal peptide (SP), is translocated onto the cell wall of B. kuwatsukai and secreted into the apoplast space of plant cells as expressed in the leaves of Nicotiana benthamiana, which can behave as a microbe-associated molecular pattern (MAMP) to trigger the defense response of plants, including cell death, reactive oxygen species (ROS) burst, callose deposition, and immunity-related genes up-regulated. It supports the conclusion that BkLiP1 plays an important role in the virulence and vegetative growth of B. kuwatsukai and alternatively behaves as an MAMP to induce plant cell death used for the fungal version, which contributes to a better understanding of the pathogenetic mechanism of Botryosphaeria fungi.
Transgenic mice encoding modern imaging probes: Properties and applications
Article
Full-text available
  • May 2022
Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.
Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells
Article
  • Apr 2022
Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.
Boosting targeted genome editing using the hei-tag
Article
Full-text available
Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straight-forward purification. Immediate activity is usually granted by administration of pre-assembled protein/RNA complexes. We present the 'hei-tag ( h igh e ff i ciency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.
CYTOPLASMIC SYNTHESIS OF NUCLEAR PROTEINS
Article
Full-text available
  • Dec 1971
The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.
Biological activity of purified SV40 T antigen proteins
Article
Full-text available
  • Apr 1978
Proteins related to simian virus 40 (SV40) T antigen uere isolated from cells infected with adenovirus 2/SV40 hybrids Ad2+D2 and Ad2+ND1 dp2 as well as from a line of human cells (SV80) transformed by SV40. The 96,000- and 107,000-dalton proteins of SV80 and Ad2+D2, after injection into the cytoplasm of cultured cells, rapidly accumulate in the nuclei, where they remain antigenically reactive for at least 20 hr and trigger DNA synthesis in quiescent cells. By contrast, the 23,000-dalton protein coded by Ad2+ND1 dp2 does not stimulate cellular DNA synthesis. However, all three purified proteins are able to provide helper function for the growth of adenovirus 2 in monkey cells. Thus, purified SV40 T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.
The Molecular Biology of Tumor Virus
Article
  • Jan 1973
Physicochemical and antigenic properties of synthetic fragments of human leukocyte interferon
Article
  • Nov 1981
Although interferons (IFNs) were originally described as proteins conferring antiviral resistance to eukaryotic cells1, various additional biological effects have since been attributed to them, including inhibition of cell proliferation and modulation of the immune response2. More recently it was recognized that interferons from different sources have similar amino acid sequences, suggesting their evolutionary relationship. When the sequences of human interferons of the α and β type were aligned to give maximum homology, a largely conserved region was found near the carboxyl terminus of the molecules3,4. The homology of human leukocyte IFN-α 1 and human fibroblast interferon between positions 111 and 166, for instance, was 40%. As conservation of segments of polypeptide chains during evolution may indicate their functional importance5, we synthesized three carboxyl-terminal fragments of human IFN-α 1 (ref. 6) ranging in size from 33 to 96 amino acid residues. If the folding of these fragments was similar to that of the corresponding segments in the intact protein, it was conceivable that they had biological activity and that antibodies raised against the synthetic fragments cross-reacted with natural interferon. Such antibodies could be used for the affinity purification of human interferon produced in bacteria and for radioimmunoassays. As we show here, none of the fragments had antiviral activity. A mouse monoclonal antibody prepared against a 56-residue fragment also bound intact IFN-α 1 and IFN-α 2 without neutralizing their antiviral effect. Thus, this antibody, directed against a largely conserved region, may show substantial cross-reactivity within the interferon protein family.
Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relation to the other avian and mammalian isoenzymes
Article
  • May 1975
Nucleocytoplasmic segregation of proteins and RNAs
Article
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Identification of simian virus 40 protein A
Article
  • Mar 1977
A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.
The amino acid sequence of β-Galactosidase of Escherichia coli
Article
  • May 1977
The amino acid sequence of beta-galactosidase was determined. The protein contains 1021 amino acid residues in a single polypeptide chain. The subunit molecular weight calculated from the sequence is 116,248. The sequence determination, carried out mainly by conventional methods, was aided by complementation tests, by the use of termination mutant strains, and by a new immunochemical method. The five residue sequence Thr-Pro-His-Pro-Ala appears twice within the polypeptide chain, but no other striking homologous features are evident.
Crystal structure of cat muscle Pyruvate Kinase at a resolution of 2.6 Å
Article
  • Nov 1979
The structure of pyruvate kinase (EC 2.7.1.40) has been determined from a 2.6 Å resolution electron density map. This map shows more detail than the previous 3.1 Å map (Stammers & Muirhead, 1977) and has enabled a detailed chain folding to be established for two out of the three domains which make up each of the four identical subunits. A provisional chain folding has been established for the third domain. The results have been briefly reported in a previous paper (Levine et al., 1978). Details of the structure determination and a further discussion of the results are presented in this paper.
Intracellular migration of nuclear proteins in Xenopus oocytes
Article
  • Apr 1978
THE protein composition of the amphibian oocyte nucleus (germinal vesicle, or GV) is very different from that of the cytoplasm, as shown by SDS electrophoresis1-3. The oocyte nucleus is known to accumulate components such as RNA polymerase4, histones5 and factors that protect DNA from nucleases6, probably stockpiled for use during early development. Furthermore, some nuclear proteins accumulate in the germinal vesicle after microinjection into the oocyte cytoplasm, as shown by Gurdon7 for labelled histones and by Bonner8, who injected labelled total GV contents into oocytes. We describe here a study of these phenomena using a recently described high resolution method of protein analysis involving two-dimensional gel electrophoresis9. Having defined those proteins which are nuclear, cytoplasmic, or present in both cell compartments of Xenopus laevis oocytes, we micro-injected a soluble preparation of labelled GV proteins into oocyte cytoplasm and analysed their distribution in the nuclear and cytoplasmic compartments. The results show that cells have exquisitely selective mechanisms that determine the extent to which a protein will become concentrated in the nucleus. Our findings suggest that nuclear proteins contain in their molecular structure a signal that enables them to accumulate in the nucleus. This signal is absent in proteins such as actin, which are similarly abundant in both nucleus and cytoplasm.