Chapter4· Viscussion

Discussion I 96

Leishmania are protozoan parasites from the family Trypanosomatidae that

cause leishmaniasis. This disease represents a major public health risk in many

tropical and subtropical regions of the world. It currently affects an estimated 12

million people in 88 countries, with approximately two million new cases

appearing every year (www.who.int/ctd/html/leis.html). The recent epidemic of

visceral leishmaniasis in the Sudan alone claimed an estimated 1, 00,000 lives

(Seaman et al, 1996). It is therefore very much desired to develop newer

methods to combat this disease as most of the existing drugs are either

ineffective against the parasite due to drug resistance or are unaffordable to the

patients. To develop more effective and less toxic newer anti-leishmania! drugs, it

is required to explore the molecular cell biology of the parasite in order to identify

the parasite proteins that are essential for its survival.

The cell morphology in most eukaryotic cells is regulated by the actin network

that underlies the plasma membrane bilayer. In association with numerous

binding proteins, actin filaments impart structural characteristics to diverse cell

types (Sheterline and Sparrow, 1994). The shape changes during various

biological processes like amoeboid movements of protozoa and human white

blood cells involve active participation of actin network that pushes forward the

plasma membrane (Pollard et al, 2001 ). However, unlike other eukaryotes, the

shape of trypanosomes is defined by an internal microtubule cytoskeleton (Gull,

1999). These microtubules are cross-linked both to each other and to the inner

face of the plasma membrane, and. have a defined polarity with their plus ends

towards the posterior of the cell (Robinson et al, 1995).

Ben Amar (1988) for the first time reported two actin gene copies tendemly

present in Trypanosoma brucei. Applying immunochemical methods, Mortara

(1989) showed the presence of actin protein in various trypanosomatids including

Leishmania maxicana using anti rabbit-actin antibodies. However, the same

Discussion I 97

could not be reproduced in T. brucei by Shi et al (2000). Actin gene from L. major

was cloned and mRNA expression level was detected in the promastigote forms

by De Arruda et al ( 1994 ). But no serious attempts were made to identify actin

expression at the protein level which may perhaps be attributed to the inability of

anti-human actin antibodies or rhodamine-phalloidin to reproducibly stain the

Leishmania actin. Most of the investigators working with Leishmania thus

preconceived for the absence of actin protein in this parasite, and the studies of

tubulins as a cytoskeletal protein prevailed in these cells. This prejudice with

actin was sustained until the DNA sequencing of kinetoplastid genomes opened

the catalog of various eukaryotic actin network associated proteins. Especially,

during L. major genome sequencing, a number of actin binding and regulating

proteins have been detected viz, actin related proteins (ARPs), coronin, profilin,

ADF/cofilin, G-actin binding proteins, myosins, formins and dynamin. The

genomic load of these many proteins is certainly indicative for the existence of

elaborate actin network in these parasites. The present study, for the first time,

reports that about 1 06 actin .copies per cell are present in the Leishmania

promastigote, and shows that Leishmania actin significantly differs from other

eukaryotic actins, especially plasmodia, yeast and mammalian actins, in terms of

its immunogenic and filament-forming properties. Unlike mammalian or yeast

actins (Reisler, 1993; Sheterline and Sparrow, 1994), Leishmania actin does not

form arrays nor are its oligomeric forms stained by phalloidin or dissociated by

Latrunculin B or Cytochalasin D.

4.1. Bioinformatic analysis

The most interesting observation of clustered dissimilar amino acids on the leish­

actin sequence posed various obvious questions regarding the structural and

functional integrity of this molecule. The major differences between the

mammalian actins and Leishmania actin exist in the six amino acid sequences of

Discussion I 98

which three sequences, viz. aa 40-53, aa 194-200 and aa 266-281, include

partially or wholly the sites that participate in subunit-subunit contacts during the

actin oligomerization to form filamentous actin (Galkin et al, 2002; Wriggers et al,

1997) which may alter the mode of polymerization process. Further, besides

being important for the subunit-subunit contacts, the amino acid residues 40-53

is present within the DNase I binding loop (Galkin et al, 2002; Wriggers et al,

1997) which perhaps suitably accounts for inability of DNase I to bind

trypanosomatid actin (Mortara, 1989). The mutation (l255D) in the aa 262-272

region, which constitutes the hydrophobic plug (Galkin et al, 2002; Wriggers et al,

1997), in yeast actin has been reported to disrupt the hydrophobic interactions,

resulting in inhibition of actin polymerisation at low temperature (Chen et al,

1993). Amino acid changes in this region in Leishmania actin are apparently

homologous (L266I) however, the physical constrains lent by the diverged flanking

region of this residue may be important. A minor but interesting observation was

a gap introduced at position 237 and an insertion of proline residue at position

274 which might bring about a structural shift in the whole molecule.

Certain structural features could be analysed by the homology model of leish­

actin. The overall sequence seems to preserve the core structure of the molecule

except the domain 2 (DNase I binding loop) whereas all the diverged stretches,

lying on the surface might give rise to changed surface topology. Since in vivo,

actin alone does not form the microfilaments and is always associated with

various binding partners, the modified surface would have changed the

interaction patterns of various actin binding proteins in Leishmania parasites.

However, it would have created organism specific adaptations of actin binding

proteins for the unique interaction strategies giving rise to various actin

assemblies. Such alterations in protein assemblies can not be generalized to the

whole family of trypanosomatids. Since Trypanosoma actin possesses -11%

Discussion I 99

sequence variation with leish-actin, structural dissimilarity may also appear even

between these two sister-parasites. Another discrepancy between these

parasites is the number of actin genes. Leishmania cells appear to have only one

actin gene, as compared to two or more genes of this protein present in the

Trypanosoma genome (de Arruda et al, 1994). This may further find support from

the various studies reported earlier on ciliates. The ciliate actins, like the

Leishmania actin, are not stained by phalloidin or recognised by antibodies

raised against other eukaryotic actins, and contain diverged amino acids in the .

DNase I binding loop (aa 40-50) as well as aa 194-200 region (Villalobo et al,

2001). Further, the primary structure and the gene copy number of these actins

also vary within this family of organisms (Villa lobo et al, 2001 ). Interestingly,

actin's structural homolog MreB in prokaryotes (Vitold et al, 2002) showed

complete absence of the corresponding sequences diverged in trypanosomatid

actin. Certainly, this may be associated with the adaptive evolution of these

organisms from their prokaryotic ancestors.

Another striking feature of the actin through sequence was the diverged

adenosine ring binding motif in this molecule. Adenosine ring is a part of ATP

molecule, which takes part in the polymerization cycle of the actin and is tightly

associated with the protein scaffold through three adenosine ring binding and two

phosphate binding motifs (Sheterline and Sparrow, 1994). After incorporation of

ATP-actin monomer into the filament, ATP hydrolyses to ADP by releasing Pi

from the molecule. This nucleotide is then swapped with ATP again after actin

depolymerisation from pointed end of the filaments, the phenomenon assisted by

ADF/cofilin and profilin (Pollard et al, 2001 ). The kinetics of this changeover is

therefore directly related to the motifs that hold the ATP/ADP molecules in the

protein. One altered adenosine ring binding motif could thus bring about altered

kinetics of ADP to ATP changeover and thereby altered microfilament dynamics,

Discussion I 100

which may be crucial for the parasite physiology. However, this motif in the actin

molecule has highest diversity than the other ATP-binding motifs throughout the

animal and plant kingdom and might be associated with the functional diversity or

stability of the different actin isotypes.

4.2. Antigenic attributes

Since diverged amino acids were clustered on leish-actin sequence, high

antigenic response through these heterologous sequences would have conferred

antigenicity to this protein. Antibody titers of both the human actin and leish-actin

showed marked differences in their antigenicity and specific recognition of actin

in Leishmania is therefore attributed to the divergent sequences on the protein

itself. However, the results of highly specific recognition of leish-actin, raised an

obvious doubt that- why L-act abs did not detect any corresponding protein in

other organisms when antigen used for raising antibodies was whole protein and

not the only diverged sequences? Through another experiment this was

confirmed that these antibodies also recognize human actin when high amount of

L-act abs (1 Oj..lg/ml) were used. It is therefore inferred that, concentration of

antibodies against .~)verged sequences (L-act absD) was perhaps significantly

greater than the antibodies against £Onserved regions (L-act absC). When this

mixed population of antibodies was applied at a high dilution (0.1 J..lg/ml),

concentration of L-act absC became too less to detect their corresponding

epitopes on the actin of other organisms by western blotting. This may suitably

explain the failure of other investigators (Shi et al, 2003) in repeating the earlier

study where rabbit actin antibodies have been used as a probe to detect actin in

Leishmania parasites (Mortara RA, 1989).

Discussion /101

4.3. Actin cytoskeleton and drug sensitivity

Actin is a major cytoskeleton protein in eukaryotes and detergent insoluble

fraction retains around 45-50% of total actin present in the cell (Joan et al, 1984).

However, trypanosomatid parasites contain tubulins as the major cytoskeletal

proteins and our results suggest that actin corresponds to only 30% of the total

cellular actin in the cytoskeleton. Reduced cytoskeletal retention of leish-actin as

compared to mammalian actin can be ascribed either as its inherent property or

presence I absence of the binding partners that stabilize I destabilize actin

cytoskeleton. Since actin cytoskeleton is regulated by various actin binding

proteins, it is a matter of debate whether catalog of actin binding proteins is

different in Leishmania as compared to mammalian cells. Analyses of leish-actin

expression in a heterologous mammalian expression system where actin binding

proteins were common for both the mammalian and Leishmania actins revealed

that leish-actin was not incorporated in mammalian actin cytoskeleton. Instead, it

formed oligomers at its own corresponding to about 30% of the total expressed

leish-actin. This further confirmed that Leishmania actin is perhaps functionally

and structurally different than the mammalian actin. Although, high actin

monomer pool is indicative of more dynamic activities of actin cytoskeleton, it is

difficult to quantitate mass of the dynamic cytoskeleton because biochemical

extraction procedures are harsh enough to set aside the dynamic mass

disassembled into soluble fraction. Thus it was likely that leish-actin existed more

as dynamic assemblies, than the biochemically defined cytoskeleton. Cell

morphology in mammalian cells is regulated by actin stress fibers spanning the

cell cytoplasm, connected with the focal adhesion points at the extracellular

matrix (Schmidt and Hall, 1998). However, leish-actin that resists latrunculin B

treatment did not form separate stress fibers and failed to maintain the

morphology of the latrunculin B-treated mammalian cells transfected with liesh-

Discussion I 102

actin gene. This is perhaps due to tendency of leish-actin to form short filam~nts

rather than the long arrays that are formed by mammalian actin. This is well

supported by the earlier study (Shi et al, 2003) where depletion of tubulin by

RNAi, transformed T.brucei cells from slender to "fat cells" whereas depletion of

actin did not affect the parasite morphology ..

4.4. Subcellular distribution

In Leishmania cells, actin was localized in the cytoplasmic compartment,

flagella, nucleus and kinetoplast. Since this protein is a globular protein, it is

capable of diffusing everywhere in the cells including nucleus. Further,

localisation of actin in the flagella may be related to the classical functioning for

whiplash action in association with light chains of dynein inner arm connected to

the flagellar microtubules (Yanagisawa and Kamiya, 2001 ). As actin in flagella of

Leishmania promastigotes was localised at the outer face of the flagellar

axoneme, it may be speculated that this protein may be performing a similar

function in Leishmania cells. Presence of actin at the microtubular corset was an

interesting observation. There are several evidences which suggest that many

fundamental cellular processes associated with cell motility, growth and

cytokinesis are governed by cross talks between microtubule and actin filaments

which maintain dynamic cellular asymmetries, polarity and cell morphology

(Goode et al, 2000). Both Immune-precipitation and confocal microscopy have

provided strong evidence for the interaction between these two cytoskeletal

components. However, interactions between actin and tubulin as small

assemblies in the cytoplasm are difficult to explain without identifying their

functions. Nevertheless, in light of the published literature, it may be suggested

that these assemblies may have some role in intracellular streaming and

vesicular transport.

Discussion I 103

Characteristic slender shape of trypanosomatid parasites is thought to be

controlled by the microtubules (Gull, 1999), and shape transformation during

growth and multiplication of these parasites in macrophages has been attributed

altered microtubular patterns and angles (Stinson et al, 1989). However, the

underlying mechanisms that regulate cell shape during cell division as well as in

the promastigote - amastigote transformations in Leishmania parasites are not

yet clear. Although, the microtubules provide scaffold for the characteristic

morphology but the force that changes their arrangement and alignment during

cell shape changes is not known. Since cortical actin is associated with the

microtubular corset in Leishmania, it is suggested that actin may perhaps

regulate the microtubule dynamics in association with some motor proteins which

ultimately results in generation of force for the cytoskeletal rearrangements.

It was interesting to note that Leishmania actin besides being localised in the

cortical region, cytosol and flagella was also present in the nucleus and

kinetoplast. Presence of actin in the nucleus has been reported in several other

eukaryotic cells (Pederson and Aebi, 2003). But up till recently, it was mainly

ascribed to its thermodynamic equilibrium (Rando et al, 2000). Scepticism on the

existence of actin in the nucleus is, however now giving way to the productive

investigations of its functional roles. And identification of actin in several nuclear

complexes implicates it in diverse nuclear activities including transcription,

chromatin remodeling and nucleo-cytoplasmic trafficking. Though the role of actin

in RNA transcription (Scheer et al, 1984) and export (Hofmann et al, 2001) had

already been hypothesized, actin along with myosin I has been reported only

recently to interact with RNA polymerase I facilitating the transcription process

(Philimonenko et al, 2004). There is compelling evidence to suggest that actin in

the nucleus exists in form of some yet undefined structures and not in the

monomeric form (Rando et al, 2000; Pederson et al, 2003; Bettinger et al, 2004),

Discussion I 104

which is further corroborated by the present study. As actin oligomerization

requires an involvement of specific actin-binding proteins (Dos Remedios et at al,

2003; Pederson et al, 2003), it is certain that Leishmania genome contains genes

that endocde actin-binding proteins required for retaining actin within the nucleus.

Potential role of ARPs have recently been implicated in chromatin remodeling

(Oiave et al, 2002). In the present study also, we have observed association of

actin with nuclear and kinetoplast DNA in both the intact cells and cell ghosts

prepared by NP-40 treatment. It was further observed that actin in nucleus was

largely present in filament-like forms rather than the granular form. Given the fact

that Leishmania actin contains two amino acid sequences, viz. aa 171-182 and

aa 212-223 that are similar to the two nuclear export signals identified earlier in

the mammalian and yeast actins (Wada et al, 1998), it is inferred that Leishmania

cells must contain some actin-binding proteins which perhaps enable the parasite

to retain actin specifically within their nuclei. Furthermore, detailed study using

immuno-gold electron microscopy revealed that Leishmania nuclear actin is

perhaps required in the nuclear division process. Presence of high amounts of

actin in the promastigotes in early prophase as well as association of this protein

with chromatin material further suggests that this protein may be involved in

chromatin condensation during the cell division process.

Three types of mitosis has been reported- 1) Open: that occurs in higher

eukaryotes, where at the onset of prophase, nuclear envelope disassembles and

reforms at the telophase stage, 2) Closed: where nuclear envelope partly

disintegrates at the prophase stage, and 3) semi-closed: where nuclear envelope

disintegrates from poles only at the metaphase stage (Kiseleva et al, 2001 ) ..

Although, the formation of intra-nuclear spindle fiber has earlier been reported in

Leishmania (Urena, 1986, 1988), but little is known about the type of the mitosis

process that this prarasite follows. Further, virtually nothing is known whether or

Discussion I 105

not actin network plays any role in this process. This apart, the factors that

preserve the structural integrity of the partially disintegrated nuclear envelope in

telophase during karyokinesis are not yet discovered. Genetic information

available from genome sequencing indicates an absence of any protein that may

constitute nucleo-skeleton in trypanosomatids, especially in Leishmania.

However, nuclear localization of a subset of actin related proteins (ARPs) whose

members profoundly influence the amount of branching of actin filaments, has

been reported in the genome of L. major. It is mentioned that the interphase

chromosomes create a curvilinear, sinusoidal inter-chromatin space (Pederson,

2000). F-actin with a persistence length of typically 3-51Jm (Steinmetz et al,

1997), would not traject very far in the inter-chromatin space without hitting a wall

of chromatin. Moreover, during chromatin condensation, the inter-chromatin

spaces will be further reduced and thereby length of actin oligomers should also

be reduced. Detailed thermodynamic studies have revealed that short actin

filaments are metastable relative to long ones, bacause at these short lengths an

incoming actin monomer's affinity for the filament is not much higher than that for

another actin monomer (Howard, 2001 ). Therefore, it is conceivable that in a

nuclear context actin predominantly forms distinct short oligomers rather than the

long filamentous polymers, which was consistent with our present findings.

Presence of myosin proteins in the nucleus of eukaryotic cells has also been

reported recently (Nowak et al, 1997; Pestic-Dragovich et al, 2000), and based

on these studies, a role of acto-myosin complex in the nuclear function has been

envisioned. Certainly, actin alone can not generate sufficient force that is

required for holding actin within the nucleus, despite its containing the nuclear

export signals. It is therefore likely that nuclear actin must be acting in

association with myosin or homologous proteins in the chromatin condensation

process.

Discussion I 106

Localisation of actin into the kinetoplast is, however, somewhat difficult to

discuss because kinetoplast is a membrane bound organelle and a protein to be

translocated into such a compartment, requires either translocation signals on

the protein itself or translocase on the kinetoplast membrane. Alternatively, it is

possible that kinetoplast may contain its own genes that transcribe for actin and

its related proteins required by this organelle. Since reports are available for the

maxi-circle encoded expression of respiratory complex proteins and several

rRNA products typical to the kinetoplast (Lukes et al, 2002) possibility for the

copy of actin gene in this organelle can not be ruled out.

The kDNA network is a highly dynamic structure, with topological interlocks

between the mini- and maxi-circles and significant network remodeling takes

place during and after replication (Klingbeil et al, 2001). Moreover, as the

replication proceeds, the number of minicircles in the network grows and the

minicircle valence rises from 3 to 6 in a process termed network remodeling

(Chen et al, 1995). During these processes, role of cytoskeleta/ structures has

been hypothesized (Michele et al, 2001 ). It is suggested that actin network

perhaps plays a central role in kDNA remodeling.

4.5. Filamentous structures and organisation of

actin

Actin is one of the main building blocks of the microfilaments in eukaryotic

cells. Purified actin has been shown to polymerize by itself under specific

conditions or in presence of nucleator proteins in vitro. However, oligomeric actin

within the cells is associated with many actin binding proteins (ABPs) that

stabilize filaments, alter filament half life, mediate interactions with other cellular

components or bring about quaternary structural assemblies of the

microfilaments (Dos Remedios et al, 2003). Filamentous structures ranging from

Discussion I 107

2-101Jm in lengths were observed in some of the Leishmania promastigotes by

immuno-fluorescence microscopy. These filaments were mostly localised close

to the sub-pellicular microtubules, and sometimes were bifurcated at the ends

and diverted towards cytoplasm. Transmission electron microscopy of ultrathin

section of whole Leishmania promastigotes and amastigotes revealed ultra­

structural view of the filamentous structures. Close proximity and micro-fibrillar

connections with the sub-pellicular microtubules is suggestive of synergy created

between these two cytoskeletal components to bring about dynamic cytoskeletal

rearrangements in Leishmania. Organisation of these microfilaments was altered

during transformation of promastigotes to amastigote, but connections with the

sub-pellicular microtubule still persisted. In trypanosomatid parasites, cell

morphology has been argued to be governed by the microtubular corset (Gull,

1999), however, associations of actin-microfilaments with the microtubular corset

can not be considered only ornamental. Though the comprehensive significance

of such structural transformation of microfilaments can not be understood at the

moment, it may be comprehended that the dynamics of the microtubules is

perhaps regulated by the actin microfilaments during the cell shape

transformation.

In the cytoskeletal preparations of Leishmania however, filamentous

structures were not observed though they retained actin at the microtubular

corset, further confirming a cross talk between actin and microtubules in these

cells. As perceived by western blotting, these samples showed -30% retention of

actin in cytoskeleton which microscopically corresponded to the fraction

associated with the microtubular corset only. Interestingly, long filamentous

structures seen in the whole cells disappeared when classical cytoskeleton

extraction methods were applied, leaving behind only the actin that was

associated with the cortical microtubules. Since actin has been reported to exist

Discussion I 108

in various non-classical forms also e.g., sheets, tubes, folded ribbons, etc.,

(Millonig et al, 1988, Steinmetz et al 1997b; Steinmetz et al, 1998; Steinmetz et

al, 1997a), it is difficult to conclude the form of actin associated with microtubules

in Leishmania cells. Filamentous actin in these cells can therefore be divided into

two subsets, -1 ), microtubule associated stable F-actin, and -2), highly

metastable cytosolic F-actin. Thus, the definition of the cytoskeleton in

Leishmania becomes more complex and elaborate.

About 106 molecules of actin appeared to be present in a Leishmania

promastigote of which about 30% are associated with the microtubular corset.

Size of Leishmania cell body is about 6-81-Jm length and an average actin filament

length appears to be 1 01-Jm (51-Jm double stranded). This length would engage

only -3500 molecules which is only 0.5% of total available actin molecules

(7X 1 05). Much higher percentage of monomeric actin pool thus indicates the

rapid assembling and disassembling capacity of leish-actin which reflects highly

dynamic nature of actin network in these cells.

Leishmania genome sequencing has revealed the presence of genes for

proteins associated with actin nucleation and branching (Formins and ARPs) but,

no protein homolog that stabilize actin filaments in the mammalian cells

(e.g.,Tropomyosin or Troponin) are detected. On the other hand, genes for actin

network regulating proteins, like ADF/cofilin, profilin and G-actin binding proteins,

have been noticed. Genomic constitution of the Leishmania parasite has

therefore ample ground to support dynamic behavior of leish-actin network.