Over the last years, the perception about the functions of CB has changed considerably. Recently found evidences assign to this 'lysosomal peptidase' key positions in cardinal processes also outside the lysosomes, like in apoptosis or cancer. Technical advances in microscopy and the development of stable chemical and genetic markers for organelles and molecules now facilitate powerful and direct in vivo approaches. They permit not only the localization of certain proteins but also the investigation of their intracellular transport, their interaction with other proteins, and their enzymatic activities, as well as the study of the cellular response in respect to overexpression or silencing of specific proteins.
In vivo, both normal tissues and especially tumours contain a population of truncated CB, which can be traced to alternative splicing (Fig 1A). Since expression and transport of CB are frequently altered in transformed and malignant cells as well as in cells undergoing apoptotic processes, we gave our attention to the investigation of such CB aberrations. For this purpose, we have labelled several recombinant CB forms by fluorescent proteins and subjected them to living cell imaging by advanced digital microscopy techniques.
Truncated cathepsin B forms
The naturally truncated Δ51CB lacks the complete signal sequence as well as parts of the N-terminal proregion. This product is barred from entering the ER, and thus from further processing and transport by the mannose-6-℗ pathway. However, the residual propeptide contains a MTS which becomes efficient and directs the predominant amount of the respective product to mitochondria . Our own observations confirm this finding. Δ51CB is expressed both in vitro and in vivo as entire 35 kDa product . This corresponds to our findings of further truncated artificial CB sequences irrespective of their size or tagging with markers: all constructs remained intact and they were not cleaved posttranslationally. Earlier assumptions  about the possible CB-specific enzymatic activity of Δ51CB were recently questioned [25, 26].
Obviously, the propeptide is indispensable for proper in vivo-folding of the mature enzyme with the typical CB activity. Interestingly, a splicing variant of cathepsin L devoid of the signal peptide also appears associated with the nucleus and exhibits a specific cleavage activity . Therefore, one should take into consideration that the truncated form(s) of CB might have cleaving characteristics, which do not become evident in the standard assays. In this report, we prove that neither the completeness of the sequence nor the CB specific enzymatic activity is relevant to the observed nuclear accumulation and induction of cell death.
Unlike CB(FLM), which is targeted to the lysosomes via ER and Golgi and partly secreted into the extracellular medium, the cytosol-expressed Δ51CB is mainly addressed to the mitochondria . Our own experiments with the same construct confirm these findings. However, deduced from our measurements, a non-negligible fraction of the expression product can also be found in the nucleoplasm. Inspection of the published data  does not contradict our findings. Nuclear fluorescence cannot arise from unspecific decay or cleavage products inasmuch as double-tagged constructs reveal similar results as the single-tagged ones indicating the integrity of the constructs. Further, we propose a targeting signal downstream of the MTS which alternatively may direct CB and derivatives thereof into the nucleus. Obviously, a hierarchy of signals encoded within the CB polypeptide determines its intracellular distribution pattern. The signal peptide and the glycosylation sites are decisive for lysosomal targeting of the FLM-product. The signal peptide and the propeptide containing the MTS are removed during the maturation process. Thus, the nuclear targeting signal might become active after release of the enzyme from lysosomes into the cytosol. In case of the truncated Δ51CB, the MTS is predominant, whereas for the artificially truncated CB forms the nuclear targeting signal is characteristic. In the past, CB was also found in cell nuclei of tumour cells and normal tissues [31, 44, 45], but until now there are almost no indications to a potential transport mechanism or a specific function. Especially in apoptotic processes, CB and other CB-like peptidases were detected also in the cell nucleus [8, 32]. However, these studies still miss a thorough scrutiny for the nuclear localization.
Here, artificially truncated CB-GFP chimaeras were used, from which the Δ72CB-construct came closest to the splicing variant Δ51CB in respect to its size. However, it was devoid of the functional sequence present in Δ51CB that encodes the N-terminal MTS. Not only Δ72CB but also the slightly shorter CB(SC) and other considerably shorter CB fragments were first expressed cytoplasmically as expected. In the sequel, they were enriched within granular structures, which were not consistent with lysosomes or mitochondria as might be supposed. Furthermore, these polypeptides were clearly proved in the nucleoplasm of several cell types. Frequently, nucleoli showed discrete regions of labelling. In contrast to the nucleus, which can be entered by both active and passive transport, the nucleoli are addressed exclusively by interaction with nucleolar building blocks . Immunocytochemistry of CB(SC), which was tagged by a myc-epitope, confirms the results of the GFP-tagging. Though, a slightly higher reticular signal distribution was observed. In addition, this proves that in spite of its size, the fluorescent protein does not sufficiently affect the affinity of CB polypeptides to the respective localization sites.
Based on these results, the capacity of cathepsins particularly in the context of nuclear localization has to be reconsidered .
There are no clues to an already known NLS in CB according to literature and to our own computational analysis. The findings suggest that the complex differential distribution of artificially truncated CB might depend on distinct targeting signals. To identify the region(s) of a potential nuclear localization signal sequence or a signal patch, respectively, a number of mutated GFP-tagged constructs were produced. Despite the elimination of extensive sequence regions, partly including potential stabilizing elements such as disulfide bridges – e.g. in CB([C211_I243del]SC) –, the specific localization persisted to a high degree. The participation of the CB light chain in this sorting procedure was excluded. The heavy chain determines the nuclear localization only; the region with the highest impact on the specific localization could be narrowed down to its C-terminal subunit. Although constructs smaller than CB(C'1) did not reveal unequivocal results, the smallest of them, CB(C'4), was not targeted specifically. The assumption that the nuclear affinity essentially depends on the prominent acidic and polar surface residue E273, which is found within the relevant region, could not be proved by several specific mutations. Excision of the differential part of CB(C'3) and CB(C'4) did also not affect the localization. The deletion did not imply adjacent residues around the active site H278 in CB(C'4), which also might be important. Hence, the results do not support the existence of a linear signal sequence. Rather a composed signal patch is likely, which evolves from the three-dimensional conformation of the polypeptide. In contrast to linear signals, such signal patches are difficult to identify exactly.
The appearance of CB(SC) and other artificially truncated constructs in the midbody is striking (Fig 4C–G). According to a recent study , midbodies have a complex composition. However, only a few peptidases and no cysteine peptidases at all were found therein. Nevertheless, the association of CB constructs with the midbody supports their nuclear occurrence.
Transport mechanisms and interaction with the nuclear matrix
The exclusion limit for free diffusing molecules through the nuclear pores is at ~60 kDa. By applying constructs well above the exclusion limit (~84 kDa) we ruled out passive transport across the nuclear pore complex with high probability. The integrity of the products was proved by immunoblotting and by FRET analysis. In the time-lapse experiments, we noticed a directional transport of granules from across the cells to the Golgi and into the nucleus without any delay at the nuclear envelope. These observations ask for a specific transport system to which the expression product might be hooked into.
Are the imported artificial CB variants possibly retained inside the nucleus because of a specific affinity to nuclear components? To answer this question, a comparative TPM-photobleaching approach was applied. The mobility of CB(SC)-EGFP and ECFP-CB(SC)-EYFP was analysed in living cells by continuous photobleaching and FRAP. We chose the freely diffusing EGFP  and the tightly chromatin-bound H2A-EGFP  as limiting controls and TIF1A-EGFP as further control with partial mobile and immobile fractions. In both technical variants of the approach, the EGFP measurements obeyed curve shapes characteristic of free diffusion (almost exclusively mobile fraction); in contrast, those of H2A-EGFP were typical for predominantly immobile molecules. Hence, both controls reacted as to be expected and in analogy to former studies [49, 50]. The courses of the CB(SC) graphs reflect an intermediate status indicating low immobile and high mobile fractions evolving from limited diffusion inside the nucleus. From this we assume that the artificially truncated CB is able to associate with nuclear matrix components. This nuclear affinity might be transferred to the naturally truncated Δ51CB form. Chromatin as a conceivable partner of interaction could be excluded from considerations by an OPM double labelling experiment of cells using GFP-tagged histone H2A and CB(SC) inasmuch as no colocalization could be observed. The relatively high amount of mobile CB(SC) might depend on scarcity of interaction partners: we have to consider that the CB products are overexpressed, other than their possible counterparts.
It was reported that the naturally truncated Δ51CB was directed to the mitochondria and that the cells died after fragmentation of the nucleus ; our observations confirm these findings. We suppose that nuclear targeting of Δ51CB might be overwhelmed by the present MTS.
Removal of this sequence in the artificially truncated Δ72CB and in further modified constructs results in their nuclear targeting and accumulation. Both overexpressed natural and artificial constructs lead to the same consequence, namely nuclear fragmentation and cell death. Neither we nor others  could prove that the induced cell death arises from apoptosis.
It was described that cell death can be preceded by a release of mature and active CB from the lysosomes and by the appearance of CB in the nucleus . Our studies of the artificially truncated constructs support these observations. Any truncated forms of CB proved to have no regular CB enzymatic activity. A proper refolding of Δ51CB to an enzymatically active form was demonstrated under in vitro conditions . However, one has also to take into consideration a different cleavage activity or functionality for the truncated variant(s) of CB. Such was recently found in case of truncated cathepsin L  and probably also cathepsin H .
The significance of results, which are obtained by overexpression, is often a contentious issue. Two arguments support the validity of our results: (i) Generally, expression levels of transfected cell populations diverge largely among individual cells. In case of CB(SC), the response to expression is severe and comprises also cells with obviously negligible expression. (ii) Time lapse video sequences demonstrate a granulation and a directed transport of CB(SC) to the nuclear region followed by fusion with the nucleus (see supplemental material).