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krit1 ( krev1 interaction trapped gene 1 ) is a gene associated with the cerebral cavernous malformation ( ccm ) disease , characterized by vascular abnormalities that cause risk of hemorrhages in the brain with consequent headache , seizures or even death .
along with krit1 , two other genes , ccm2 ( cerebral cavernous malformation 2 ) and pdcd10 ( programmed cell death 10 ) , were linked to ccm disease , all the three genes coding for scaffold proteins lacking catalytic domains but presenting several domains for protein - protein interaction . in the last years , several studies have provided new information on the signaling pathway in which krit1 takes part , suggesting a role in vascular development , cellcell junctions , and cell - extracellular matrix adhesion . however
, none of these studies was focused on the nuclear compartment where it is known krit1 can be physiologically present , as seen by fluorescence resonance energy transfer ( fret ) experiments and immunofluorescence .
indeed krit1 , together with its protein interacting partner , icap1 ( integrin beta-1-binding protein 1 ) , shuttles between the nucleus and the cytoplasm bearing a putative nls in its n - terminal amino acid sequence .
although the shuttling dynamics involving krit1 , icap1 and ccm2 is only partially understood , there is no evidence in literature of a possible active function of krit1 inside the nuclear compartment .
one of the current hypotheses is that krit1 and icap1 regulate each other by sequestering the partner inside the nucleus , thus preventing interaction with cytoplasmic proteins , such as beta-1 integrin or the p-38-mapk associated complex . by means of ultrastructural immunocytochemistry
we detected krit1 inside the nucleus with the aim to gain insight into other possible roles of this protein , as suggested by its localization on nuclear structures of known function .
the nuclear location of krit1 was investigated by transmission electron microscope in hela cells under normal growth conditions or after heat shock when the nuclear distribution of ribonucleoproteins ( rnps ) is modified , as well as in rat hepatocytes and in chironomus thummi salivary glands ( the latter being an excellent model for transcription due to the presence of actively transcribed polytenic chromosomes ) .
the results obtained suggest that krit1 is involved in the maturation of nascent rna transcripts , due to its prevalent localization in nuclear sites where active transcription takes place .
hela cells were grown in dmem medium supplemented with 10% fetal bovine serum , 2 mm glutamine and 100 units each of streptomycin and penicillin , in 25 cm plastic flasks or on 4 cm glass coverslips in multi - well dishes on air atmosphere containing 5% of co2 at 37c ( all reagents and plastics were from euroclone , milan , italy ) . for the heat shock treatment
, the cells were incubated at 42c for 1 h , and then kept at 37c for additional 3 h before fixation .
the cells were harvested by mild trypsinization ( 0.25% trypsin in pbs containing 0.5% edta ) and immediately fixed with 4% paraformaldehyde ( 2 h at 4c ) in the culture medium .
the cell pellets were embedded in 2% agar in water , thoroughly rinsed with srensen buffer ( ph 7.2 ) and dehydrated in ethanol .
finally , the cells were embedded in lr white resin and polymerized at 60c for 24 h. thin sections were collected on nickel grids covered with a formvar carbon film . for immunolabeling
, the grids were incubated on normal goat serum ( ngs ) 1:50 for 3 min and then floated for 17 h at room temperature onto a solution of 0.1% bovine serum albumine and 0.05% tween 20 in pbs containing the primary anti - krit1 antibody ( rabbit - polyclonal against human epitope of krit1 , genetex , gtx109560 ) diluted 1:5 , and eventually one of the following primary mouse - monoclonal antibody to detect co - localization : anti - pol ii ( against human heptapeptide epitope of rna polymerase ii , and recognizing both the active and inactive forms ; biodesign , m55701 m ) diluted 1:5 ; anti h20 ( recognizing the 3 mg cap of snrnas , kindly provided by dr .
after rinsing with pbs - tween and pbs , grids were incubated with ngs as above , and then reacted with a goat - anti rabbit and a goat anti - mouse secondary antibodies conjugated with 12 nm or 6 nm colloidal gold particles ( the jackson lab . , bar harbor , me , usa ) for 30 min at room temperature , rinsed and finally stained .
to reduce chromatin contrast and selectively reveal nuclear ribonucleoprotein constituents , the sections were bleached by the edta method or selectively stained for rna with terbium citrate .
the samples were observed on a zeiss 900 em operating at 80kv . in some of the micrographs ,
hela cells were grown in dmem medium supplemented with 10% fetal bovine serum , 2 mm glutamine and 100 units each of streptomycin and penicillin , in 25 cm plastic flasks or on 4 cm glass coverslips in multi - well dishes on air atmosphere containing 5% of co2 at 37c ( all reagents and plastics were from euroclone , milan , italy ) . for the heat shock treatment
, the cells were incubated at 42c for 1 h , and then kept at 37c for additional 3 h before fixation .
the cells were harvested by mild trypsinization ( 0.25% trypsin in pbs containing 0.5% edta ) and immediately fixed with 4% paraformaldehyde ( 2 h at 4c ) in the culture medium .
the cell pellets were embedded in 2% agar in water , thoroughly rinsed with srensen buffer ( ph 7.2 ) and dehydrated in ethanol .
finally , the cells were embedded in lr white resin and polymerized at 60c for 24 h. thin sections were collected on nickel grids covered with a formvar carbon film . for immunolabeling
, the grids were incubated on normal goat serum ( ngs ) 1:50 for 3 min and then floated for 17 h at room temperature onto a solution of 0.1% bovine serum albumine and 0.05% tween 20 in pbs containing the primary anti - krit1 antibody ( rabbit - polyclonal against human epitope of krit1 , genetex , gtx109560 ) diluted 1:5 , and eventually one of the following primary mouse - monoclonal antibody to detect co - localization : anti - pol ii ( against human heptapeptide epitope of rna polymerase ii , and recognizing both the active and inactive forms ; biodesign , m55701 m ) diluted 1:5 ; anti h20 ( recognizing the 3 mg cap of snrnas , kindly provided by dr .
, grids were incubated with ngs as above , and then reacted with a goat - anti rabbit and a goat anti - mouse secondary antibodies conjugated with 12 nm or 6 nm colloidal gold particles ( the jackson lab . , bar harbor , me , usa ) for 30 min at room temperature , rinsed and finally stained . to reduce chromatin contrast and selectively reveal nuclear ribonucleoprotein constituents , the sections were bleached by the edta method or selectively stained for rna with terbium citrate .
the samples were observed on a zeiss 900 em operating at 80kv . in some of the micrographs ,
after edta staining to reveal rnps , krit1 labeling in hela cells was localized on the perichromatin region , where transcription takes place and , in particular , on the perichromatin fibrils ( pf , the in situ morphological expression of the extranucleolar rna transcription ) ( figure 1a ; supplementary figure 1 ) .
this pattern was also observed after heat shock , suggesting that the nuclear localization of krit1 is not influenced by this type of stress .
more in detail , staining with terbium citrate for rna allowed us to better visualize the nascent pfs labeled with the krit1 antibody ( figure 1b ) .
we were able to observe the same pattern in salivary glands of chironomus thummi : fig 1c shows a pf where the labelings for rna pol ii and krit1 are present at the opposite extremities . since the antibody does not discriminate between the active and inactive form of rna pol ii , we can not determine if that pf is being transcribed , although it is tempting to assume so . in this model system , krit1 is also present in the large ( perichromatin - granules like ) balbiani granules , where the mrna transcribed is stored for the export to the cytoplasm ( figure 2a ) .
we rarely found krit1 co - localizing with rna pol ii , whereas we observed a co - localization with the methyl - cap of snrna . in figure 2b an example of double labeling in a hela cell after heat shock is presented . in all the cell types used in these experiments ,
we have observed focal electron dense areas of the nucleus that show a higher labeling for krit1 suggesting the presence of nuclear bodies of unknown - function ( figure 2 c , d ) .
these labeled nuclear domains were observed in all immunolabeling experiments in condition of minimum background , which leads us to exclude an artifactual labeling .
labeling for pol ii was never found on these nuclear domains . finally , we have documented krit1 presence in the dense fibrillar component ( dfc ) of the nucleolus in numerous hela samples ( figure 2 e , f ; supplementary figure 2 ) .
the same labeling distribution is present on the nucleolus in hepatocytes ( not shown ) .
our results provide novel information about the fine nuclear distribution of the protein krit1 and its spatial relationships with the rnp components involved in rna transcription and maturation , confirming and extending previous data on its nuclear localization . despite its known role in cell - cell junction and cell - matrix anchorage
, krit1 shows a peculiar nuclear labeling on the pfs and nucleolar dfc , where transcription actively takes place .
the recurrent presence of this labeling pattern in most of the cells in the samples observed , from human hela cells to chironomus thummi salivary glands , suggests a possible conserved function of krit1 inside the nuclear compartment , reasonably involving rna transcription .
the presence of the labeling at the terminus of the newly transcribed pfs ( i.e. , far away from pol ii but associated with the m3 g cap ) , together with the lack of dna binding domains in the krit1 protein structure , leads us to hypothesize that krit1 could be involved in rna maturation rather than be directly involved in transcription .
also the presence of krit1 labeling in the perichromatin - granules - like balbiani granules could support this idea .
no marked difference was observed in hela cells before and after heat shock , suggesting that krit1 nuclear localization and/or shuttling is not modified or increased by heat shock .
as it is the case for other nuclear bodies , their function could be related to the nucleus - cytoplasmic shuttling of krit1 .
being positive to terbium and edta staining we know they are composed of rnps , but further studies are needed to understand the nature of these nuclear domains .
these data lay the basis for a further characterization of krit1 function inside the nuclear compartment .
it is not excluded that an impairment of this unknown krit1 nuclear function could contribute to the development of ccm in the individuals that carry mutations on this gene .
furthermore , these data can be implemented by observing the nuclear labeling pattern of the ccm2 and pdcd10 protein products , involved in the development of the disease .
being the pathogenetic mechanisms by which krit1 mutation cause familial ccm still unknown , we believe that adding new information on the complex molecular pathway of these genes could lead to a better understanding of the ccm pathology .
krit1 is an 84kda protein that lacks any relevant catalytic domains , associated with the cerebral cavernous malformation disease . 
 we have investigated by means of ultrastructural immunocytochemistry the nuclear distribution of krit1 in different cell lines , revealing its unexpected localization on actively transcribing nuclear domains such as the perichromatin fibrils and the nucleolar dense fibrillar component . 
 these preliminary data indicate a still undescribed and unknown role for krit1 inside the nucleus .